Reconstitution of abnormalities in the malignant hyperthermia-susceptible pig ryanodine receptor

1993 ◽  
Vol 264 (1) ◽  
pp. C125-C135 ◽  
Author(s):  
N. H. Shomer ◽  
C. F. Louis ◽  
M. Fill ◽  
L. A. Litterer ◽  
J. R. Mickelson
Keyword(s):  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Single Channel ◽  
Ryanodine Receptors ◽  
Primary Defect ◽  
Open Probability ◽  
Release Channel ◽  
Malignant Hyperthermia Susceptible ◽  
Channel Opening ◽  
Kd Values

Malignant hyperthermia-susceptible (MHS) pigs homozygous for the Cys615 ryanodine receptor allele demonstrate altered sarcoplasmic reticulum (SR) ryanodine binding and Ca2+ release channel regulatory properties when compared with normal pigs homozygous for the Arg615 allele. While solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the purified MHS and normal ryanodine receptors had a similar dissociation constant (Kd) for ryanodine, maximum binding, and Ca2+ concentration for half-maximal stimulation and inhibition of ryanodine binding (Ca2+(0.5)); however, after reconstitution into proteoliposomes, the purified MHS and normal receptors had Kd values for ryanodine of 75 and 150 nM, respectively, which were significantly different. The purified MHS and normal porcine ryanodine receptors also had similar single-channel Cs+ conductance, optimal cis-Ca2+ for channel opening, and cis-Ca2+(0.5) for channel activation. Significantly, at inactivating levels of cis-Ca2+ (> 0.1 mM), MHS channels had a greater open probability, a higher cis-Ca2+(0.5) for inhibition of channel opening (250 vs. 75 microM for MHS and normal, respectively), longer mean open times, and shorter mean closed times than did normal channels. We conclude that the mutation at residue 615 causes a detectable alteration in ryanodine receptor/Ca2+ channel activity and thus may represent the primary defect responsible for the altered SR Ca2+ regulation characteristic of MHS porcine muscle.

Caffeine stimulation of malignant hyperthermia-susceptible sarcoplasmic reticulum Ca2+ release channel

1994 ◽  
Vol 267 (5) ◽  
pp. C1253-C1261 ◽  
Author(s):  
N. H. Shomer ◽  
J. R. Mickelson ◽  
C. F. Louis
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Malignant Hyperthermia ◽  
Single Channel ◽  
Release Channel ◽  
Muscle Contracture ◽  
Malignant Hyperthermia Susceptible ◽  
Open Time ◽  
Pig Muscle ◽  
Channel Mutation ◽  
The Difference

The altered caffeine sensitivity of malignant hyperthermia-susceptible (MHS) muscle contracture is one basis of the diagnostic test for this syndrome. To determine whether the Arg615-to-Cys615 mutation of the porcine sarcoplasmic reticulum (SR) Ca2+ release channel is directly responsible for this altered caffeine sensitivity, the single-channel kinetics of purified MHS and normal pig Ca2+ release channels were examined. Initial studies demonstrated that decreasing the pH of the medium in either the cis- or trans-chamber decreased the Ca2+ release channel percent open time (Po). The half-inhibitory pH of MHS channels (6.86 +/- 0.04, n = 17) was significantly different from that of normal channels (7.08 +/- 0.07, n = 14). At pH 7.4, in either 7 or 0.12 microM Ca2+, MHS channel Po was not significantly different from that of normal channels over the range 0-10 mM caffeine. Although at pH 6.8 in 7 microM Ca2+ MHS channel Po was greater than that of normal channels over the range 0-20 mM caffeine, the difference could be eliminated by dividing each mean MHS Po by a scaling factor of 3.2. Thus the MHS Ca2+ release channel mutation does not appear to be directly responsible for the altered caffeine sensitivity of MHS pig muscle contracture. Rather, this altered caffeine sensitivity may result from an altered resting myoplasmic Ca2+ concentration or the altered pH and Ca2+ sensitivity of Ca2+ release channel Po of MHS muscle.

scholarly journals Nicotinic acid–adenine dinucleotide phosphate activates the skeletal muscle ryanodine receptor

2002 ◽  
Vol 367 (2) ◽  
pp. 423-431 ◽  
Author(s):  
Martin HOHENEGGER ◽  
Josef SUKO ◽  
Regina GSCHEIDLINGER ◽  
Helmut DROBNY ◽  
Andreas ZIDAR
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Nicotinic Acid ◽  
Ryanodine Receptor ◽  
Spatial Information ◽  
Single Channel ◽  
Reversal Potential ◽  
Open Probability ◽  
Release Channel

Calcium is a universal second messenger. The temporal and spatial information that is encoded in Ca2+-transients drives processes as diverse as neurotransmitter secretion, axonal outgrowth, immune responses and muscle contraction. Ca2+-release from intracellular Ca2+ stores can be triggered by diffusible second messengers like InsP3, cyclic ADP-ribose or nicotinic acid—adenine dinucleotide phosphate (NAADP). A target has not yet been identified for the latter messenger. In the present study we show that nanomolar concentrations of NAADP trigger Ca2+-release from skeletal muscle sarcoplasmic reticulum. This was due to a direct action on the Ca2+-release channel/ryanodine receptor type-1, since in single channel recordings, NAADP increased the open probability of the purified channel protein. The effects of NAADP on Ca2+-release and open probability of the ryanodine receptor occurred over a similar concentration range (EC5030nM) and were specific because (i) they were blocked by Ruthenium Red and ryanodine, (ii) the precursor of NAADP, NADP, was ineffective at equimolar concentrations, (iii) NAADP did not affect the conductance and reversal potential of the ryanodine receptor. Finally, we also detected an ADP-ribosyl cyclase activity in the sarcoplasmic reticulum fraction of skeletal muscle. This enzyme was not only capable of synthesizing cyclic GDP-ribose but also NAADP, with an activity of 0.25nmol/mg/min. Thus, we conclude that NAADP is generated in the vicinity of type 1 ryanodine receptor and leads to activation of this ion channel.

scholarly journals Calmodulin sensitivity of the sarcoplasmic reticulum ryanodine receptor from normal and malignant-hyperthermia-susceptible muscle

1996 ◽  
Vol 319 (2) ◽  
pp. 421-426 ◽  
Author(s):  
Sean O'DRISCOLL ◽  
Tommie V. McCARTHY ◽  
Hans M. EICHINGER ◽  
Wolf ERHARDT ◽  
Frank LEHMANN-HORN ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Central Region ◽  
Dependent Manner ◽  
Release Channel ◽  
Malignant Hyperthermia Susceptible ◽  
Potential Binding ◽  
Cam Regulation

Ca2+ release from sarcoplasmic reticulum (SR) of malignant-hyperthermia-susceptible (MHS) muscle is hypersensitive to Ca2+ and caffeine. To determine if an abnormal calmodulin (CaM) regulation of the SR Ca2+-release-channel-ryanodine-receptor complex (RYR1) contributes to this hypersensitivity, we investigated the effect of CaM on high-affinity [3H]ryanodine binding to isolated SR vesicles from normal and MHS pig skeletal muscle. CaM modulated [3H]ryanodine binding in a Ca2+-dependent manner. In the presence of maximally activating Ca2+ concentrations, CaM inhibited [3H]ryanodine binding with no differences between normal and MHS vesicles. In the absence of Ca2+, however, CaM activated [3H]ryanodine binding with a 2-fold-higher potency in MHS vesicles. Significant differences between normal and MHS tissue were observed for CaM concentrations between 50 nM and 10 µM. A polyclonal antibody raised against the central region of RYR1 specifically inhibited this activating effect of CaM without affecting the inhibition by CaM. This indicates that the central region of RYR1 is a potential binding domain for CaM in the absence of Ca2+. It is suggested that in vivo an enhanced CaM sensitivity of RYR1 might contribute to the abnormal high release of Ca2+ from the SR of MHS muscle.

scholarly journals Chloroform extract of hog barn dust modulates skeletal muscle ryanodine receptor calcium-release channel (RyR1)

2010 ◽  
Vol 109 (3) ◽  
pp. 830-839 ◽  
Author(s):  
Chengju Tian ◽  
Chun Hong Shao ◽  
Danielle S. Fenster ◽  
Mark Mixan ◽  
Debra J. Romberger ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Ryanodine Receptor ◽  
Muscle Weakness ◽  
Calcium Release ◽  
Single Channel ◽  
Calcium Release Channel ◽  
Open Probability ◽  
Skeletal Muscle Function ◽  
Release Channel ◽  
Skeletal Muscle Weakness

Skeletal muscle weakness is a reported ailment in individuals working in commercial hog confinement facilities. To date, specific mechanisms responsible for this symptom remain undefined. The purpose of this study was to assess whether hog barn dust (HBD) contains components that are capable of binding to and modulating the activity of type 1 ryanodine receptor Ca2+-release channel (RyR1), a key regulator of skeletal muscle function. HBD collected from confinement facilities in Nebraska were extracted with chloroform, filtered, and rotary evaporated to dryness. Residues were resuspended in hexane-chloroform (20:1) and precipitates, referred to as HBDorg, were air-dried and studied further. In competition assays, HBDorg dose-dependently displaced [3H]ryanodine from binding sites on RyR1 with an IC50 of 1.5 ± 0.1 μg/ml ( Ki = 0.4 ± 0.0 μg/ml). In single-channel assays using RyR1 reconstituted into a lipid bilayer, HBDorg exhibited three distinct dose-dependent effects: first it increased the open probability of RyR1 by increasing its gating frequency and dwell time in the open state, then it induced a state of reduced conductance (55% of maximum) that was more likely to occur and persist at positive holding potentials, and finally it irreversibly closed RyR1. In differentiated C2C12 myotubes, addition of HBD triggered a rise in intracellular Ca2+ that was blocked by pretreatment with ryanodine. Since persistent activation and/or closure of RyR1 results in skeletal muscle weakness, these new data suggest that HBD is responsible, at least in part, for the muscle ailment reported by hog confinement workers.

scholarly journals Activation and labelling of the purified skeletal muscle ryanodine receptor by an oxidized ATP analogue

1995 ◽  
Vol 308 (1) ◽  
pp. 119-125 ◽  
Author(s):  
M Hohenegger ◽  
A Herrmann-Frank ◽  
M Richter ◽  
F Lehmann-Horn
Keyword(s):  
Skeletal Muscle ◽  
Ryanodine Receptor ◽  
Single Channel ◽  
Adenine Nucleotide ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Hill Coefficient ◽  
Dependent Manner ◽  
Open Probability ◽  
Release Channel

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.

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.

Activation of cardiac ryanodine receptors by the calcium channel agonist FPL-64176

2002 ◽  
Vol 283 (1) ◽  
pp. H331-H338 ◽  
Author(s):  
J. Andrew Wasserstrom ◽  
Leslie A. Wasserstrom ◽  
Andrew J. Lokuta ◽  
James E. Kelly ◽  
Sireen T. Reddy ◽  
...  
Keyword(s):  
Single Channel ◽  
Ryanodine Receptors ◽  
Channel Activity ◽  
Open Probability ◽  
Release Channel ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Significant Difference ◽  
Single Channel Activity ◽  
Control Versus ◽  
Channel Agonist

We investigated the possibility that the Ca2+ channel agonist FPL-64176 (FPL) might also activate the cardiac sarcoplasmic reticulum (SR) Ca2+ release channel ryanodine receptor (RyR). The effects of FPL were tested on single channel activity of purified and crude vesicular RyR (RyR2) isolated from human and dog hearts using the planar lipid bilayer technique. FPL (100–200 μM) increased single channel open probability ( P o) when added to the cytoplasmic side of the channel ( P o = 0.070 ± 0.021 in control RyR2; 0.378 ± 0.086 in 150 μM FPL, n = 9, P < 0.01) by prolonging open times and decreasing closed times without changing current magnitude. FPL had no effect on P o when added to the trans (luminal) side of the bilayer ( P o = 0.079 ± 0.036 in control and 0.103 ± 0.066 in FPL, n = 4, no significant difference). The bell-shaped [Ca2+] dependence of [3H]ryanodine binding and of P o was altered by FPL, suggesting that the mechanism by which FPL increases channel activity is by an increase in Ca2+-induced activation at low [Ca2+] (without a change in threshold) and suppression of Ca2+-induced inactivation at high [Ca2+]. However, the fact that inactivation was restored at elevated [Ca2+] suggests a competitive interaction between Ca2+ and FPL on inactivation. FPL had no effect on RyR skeletal channels (RyR1), where P o was 0.039 ± 0.005 in control versus 0.030 ± 0.006 in 150 μM FPL (no significant difference). These results suggest that, in addition to its ability to activate the L-type Ca2+channels, FPL activates cardiac RyR2 primarily by reducing the Ca2+ sensitivity of inactivation.

Activation of purified cardiac ryanodine receptors by dihydropyridine agonists

2001 ◽  
Vol 280 (3) ◽  
pp. H1201-H1207 ◽  
Author(s):  
Toshio Sagawa ◽  
Manabu Nishio ◽  
Kazuko Sagawa ◽  
James E. Kelly ◽  
Andrew J. Lokuta ◽  
...  
Keyword(s):  
Single Channel ◽  
Ryanodine Receptors ◽  
Channel Activity ◽  
Allosteric Site ◽  
Open Probability ◽  
Release Channel ◽  
Inotropic Effects ◽  
Cytoplasmic Face ◽  
Single Channel Activity ◽  
Negative Inotropic Effects

Prior observations have raised the possibility that dihydropyridine (DHP) agonists directly affect the sarcoplasmic reticulum (SR) cardiac Ca2+ release channel [i.e., ryanodine receptor (RyR)]. In single-channel recordings of purified canine cardiac RyR, both DHP agonists (−)-BAY K 8644 and (+)-SDZ202-791 increased the open probability of the RyR when added to the cytoplasmic face of the channel. Importantly, the DHP antagonists nifedipine and (−)-SDZ202-791 had no competitive blocking effects either alone or after channel activation with agonist. Thus there is a stereospecific effect of SDZ202-791, such that the agonist activates the channel, whereas the antagonist has little effect on channel activity. Further experiments showed that DHP agonists changed RyR activation by suppressing Ca2+-induced inactivation of the channel. We concluded that DHP agonists can also influence RyR single-channel activity directly at a unique allosteric site located on the cytoplasmic face of the channel. Similar results were obtained in human purified cardiac RyR. An implication of these data is that RyR activation by DHP agonists is likely to cause a loss of Ca2+ from the SR and to contribute to the negative inotropic effects of these agents reported by other investigators. Our results support this notion that the negative inotropic effects of DHP agonists result in part from direct alteration in the activity of RyRs.

scholarly journals Functional Characterization of a Distinct Ryanodine Receptor Mutation in Human Malignant Hyperthermia-susceptible Muscle

1997 ◽  
Vol 272 (8) ◽  
pp. 5256-5260 ◽  
Author(s):  
Michael Richter ◽  
Lothar Schleithoff ◽  
Thomas Deufel ◽  
Frank Lehmann-Horn ◽  
Annegret Herrmann-Frank
Keyword(s):  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Functional Characterization ◽  
Malignant Hyperthermia Susceptible

Ryanodine receptor dysfunction in porcine stunned myocardium

1997 ◽  
Vol 273 (2) ◽  
pp. H796-H804 ◽  
Author(s):  
C. Valdivia ◽  
J. O. Hegge ◽  
R. D. Lasley ◽  
H. H. Valdivia ◽  
R. Mentzer
Keyword(s):  
Coronary Artery ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Myocardial Stunning ◽  
Single Channel ◽  
Stunned Myocardium ◽  
Wall Thickening ◽  
Planar Lipid Bilayers ◽  
Open Probability ◽  
Single Channel Recordings

We investigated the effects of myocardial stunning on the function of the two main Ca2+ transport proteins of the sarcoplasmic reticulum (SR), the Ca(2+)-adenosinetriphosphatase and the Ca(2+)-release channel or ryanodine receptor. Regional myocardial stunning was induced in open-chest pigs (n = 6) by a 10-min occlusion of the left anterior descending coronary artery (LAD) and 2 h reperfusion. SR vesicles isolated from the LAD-perfused region (stunned) and the normal left circumflex coronary artery (LC)-perfused region were used to assess the oxalate-supported 45Ca2+ uptake, [3H]ryanodine binding, and single-channel recordings of ryanodine-sensitive Ca(2+)-release channels in planar lipid bilayers. Myocardial stunning decreased LAD systolic wall thickening to 20% of preischemic values. The rate of SR 45Ca2+ uptake in the stunned LAD bed was reduced by 37% compared with that of the normal LC bed (P < 0.05). Stunning was also associated with a 38% reduction in the maximal density of high-affinity [3H]ryanodine binding sites (P < 0.05 vs. normal LC) but had no effect on the dissociation constant. The open probability of ryanodine-sensitive Ca(2+)-release channels determined by single channel recordings in planar lipid bilayers was 26 +/- 2% for control SR (n = 33 channels from 3 animals) and 14 +/- 2% for stunned SR (n = 21 channels; P < 0.05). This depressed activity of SR function observed in postischemic myocardium could be one of the mechanisms underlying myocardial stunning.

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