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Biophysical Journal Dec 1997Changes in the intracellular free calcium concentration ([Ca2+]i) control many important processes in excitable and nonexcitable cells. In cerebellar Purkinje neurons,...
Changes in the intracellular free calcium concentration ([Ca2+]i) control many important processes in excitable and nonexcitable cells. In cerebellar Purkinje neurons, increases in [Ca2+]i modulate excitability by turning on calcium-activated potassium and chloride conductances, and modifying the synaptic efficacy of inhibitory and excitatory inputs to the cell. Calcium release from the intracellular stores plays an important role in the regulation of [Ca2+]i. Purkinje neurons contain both inositol trisphosphate (InsP3) and ryanodine (Ry) receptors. With the exception of the dendritic spines, where only InsP3 receptors are found, InsP3 and Ry receptors are present in the entire cell. The distribution of the two calcium release channels, however, is not uniform, and it has been suggested that InsP3 and Ry receptors use separate Ca2+ pools. The functional properties of InsP3 and Ry Ca2+ pools were investigated by flash photolysis and single-cell microspectrofluorimetry. It was found that depletion of ryanodine-sensitive Ca2+ stores renders InsP3 incapable of releasing more Ca2+ from the stores. Abolishing calcium-induced calcium release by blocking ryanodine receptors with ruthenium red did not have a significant effect on InsP3-evoked Ca2+ release. It is concluded that InsP3 receptors use the same functional Ca2+ pool as that utilized by Ry receptors in Purkinje neurons.
Topics: Animals; Biophysical Phenomena; Biophysics; Calcium; Cell Compartmentation; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Photolysis; Purkinje Cells; Rats; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel
PubMed: 9414245
DOI: 10.1016/S0006-3495(97)78359-0 -
Europace : European Pacing,... Jun 2019Action potential duration (APD) alternans is an established precursor or arrhythmia and sudden cardiac death. Important differences in fundamental electrophysiological...
AIMS
Action potential duration (APD) alternans is an established precursor or arrhythmia and sudden cardiac death. Important differences in fundamental electrophysiological properties relevant to arrhythmia exist between experimental models and the diseased in vivo human heart. To investigate mechanisms of APD alternans using a novel approach combining intact heart and cellular cardiac electrophysiology in human in vivo.
METHODS AND RESULTS
We developed a novel approach combining intact heart electrophysiological mapping during cardiac surgery with rapid on-site data analysis to guide myocardial biopsies for laboratory analysis, thereby linking repolarization dynamics observed at the organ level with underlying ion channel expression. Alternans-susceptible and alternans-resistant regions were identified by an incremental pacing protocol. Biopsies from these sites (n = 13) demonstrated greater RNA expression in Calsequestrin (CSQN) and Ryanodine (RyR) and ion channels underlying IK1 and Ito at alternans-susceptible sites. Electrical restitution properties (n = 7) showed no difference between alternans-susceptible and resistant sites, whereas spatial gradients of repolarization were greater in alternans-susceptible than in alternans-resistant sites (P = 0.001). The degree of histological fibrosis between alternans-susceptible and resistant sites was equivalent. Mathematical modelling of these changes indicated that both CSQN and RyR up-regulation are key determinants of APD alternans.
CONCLUSION
Combined intact heart and cellular electrophysiology show that regions of myocardium in the in vivo human heart exhibiting APD alternans are associated with greater expression of CSQN and RyR and show no difference in restitution properties compared to non-alternans regions. In silico modelling identifies up-regulation and interaction of CSQN with RyR as a major mechanism underlying APD alternans.
Topics: Action Potentials; Arrhythmias, Cardiac; Biopsy; Calsequestrin; Electrophysiologic Techniques, Cardiac; Female; Heart Conduction System; Humans; Ion Channels; Male; Middle Aged; Ryanodine
PubMed: 30753421
DOI: 10.1093/europace/euz007 -
Plant Signaling & Behavior Dec 2010The marine alga Ulva compressa (Chlorophyta) showed a triphasic release of intracellular calcium with maximal levels at 2, 3 and 12 h and a biphasic accumulation of...
The marine alga Ulva compressa (Chlorophyta) showed a triphasic release of intracellular calcium with maximal levels at 2, 3 and 12 h and a biphasic accumulation of intracellular hydrogen peroxide with peaks at 3 and 12 h when cultivated with copper excess. Intracellular hydrogen peroxide originated exclusively in organelles. In this work, we analyzed the intracellular origin of calcium release and the type of calcium channels activated in response to copper excess. U. compressa was treated with thapsigargin, an inhibitor of endoplasmic reticulum (ER) calcium ATPase, ryanodine, an inhibitor of ryanodine-sensitive channels, and xestospongin C, an inhibitor of inositol 1, 4, 5-triphosphate (IP(3))-sensitive channels. Thapsigargin induced the depletion of calcium stored in ER at 75 min and completely inhibited calcium release at 2, 3 and 12 h of copper exposure indicating that calcium release originated in ER. In addition, ryanodine and xestospogin C inhibited calcium release at 2 and 3 h of copper exposure whereas the peak at 12 h was only inhibited by ryanodine. Thus, copper induced the activation of ryanodine-sensitive and IP(3)-sensitive calcium channels in ER of U. compressa.
Topics: Calcium; Copper; Endoplasmic Reticulum; Inositol 1,4,5-Trisphosphate; Ryanodine; Ulva
PubMed: 21139437
DOI: 10.4161/psb.5.12.13977 -
Biophysical Journal Aug 1996We purified and characterized ryanotoxin, an approximately 11.4-kDa peptide from the venom of the scorpion Buthotus judiacus that induces changes in ryanodine receptors...
We purified and characterized ryanotoxin, an approximately 11.4-kDa peptide from the venom of the scorpion Buthotus judiacus that induces changes in ryanodine receptors of rabbit skeletal muscle sarcoplasmic reticulum analogous to those induced by the alkaloid ryanodine. Ryanotoxin stimulated Ca2+ release from sarcoplasmic reticulum vesicles and induced a state of reduce unit conductance with a mean duration longer than that of unmodified ryanodine receptor channels. With Cs+ as the current carrier, the slope conductance of the state induced by 1 microM ryanotoxin was 163 +/- 12 pS, that of the state induced by 1 microM ryanodine was 173 +/- 26 pS, and that of control channels was 2.3-fold larger (396 +/- 25 pS). The distribution of substate events induced by 1 microM RyTx was biexponential and was fitted with time constants approximately 10 times shorter than those fitted to the distribution of substates induced by 1 microM ryanodine. Bath-applied 5 microM ryanotoxin had no effect on the excitability of mouse myotubes in culture. When 5 microM ryanotoxin was dialyzed into the cell through the patch pipette in the whole-cell configuration, there was a voltage-dependent increase in the amplitude of intracellular Ca2+ transients elicited by depolarizing potentials in the range of -30 to +50 mV. Ryanotoxin increased the binding affinity of [3H]ryanodine in a reversible manner with a 50% effective dose (ED50) of 0.16 microM without altering the maximum number (Bmax) of [3H]ryanodine-binding sites. This result suggested that binding sites for ryanotoxin and ryanodine were different. Ryanotoxin should prove useful in identifying domains coupling the ryanodine receptor to the voltage sensor, or domains affecting the gating and conductance of the ryanodine receptor channel.
Topics: Animals; Calcium; Calcium Channels; Cells, Cultured; Cesium; Chromatography, High Pressure Liquid; Electric Conductivity; Fetus; Kinetics; Membrane Potentials; Mice; Muscle Proteins; Muscle, Skeletal; Rabbits; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Scorpion Venoms
PubMed: 8842209
DOI: 10.1016/S0006-3495(96)79270-6 -
Biophysical Journal Feb 1995Two isoforms of the sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor or RYR) are expressed together in the skeletal muscles of most vertebrates. We have...
Two isoforms of the sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor or RYR) are expressed together in the skeletal muscles of most vertebrates. We have studied physiological properties of the two isoforms (alpha and beta) by comparing SR preparations from specialized fish muscles that express the alpha isoform alone to preparations from muscles containing both alpha and beta. Regulation of channel activity was assessed through [3H]ryanodine binding and reconstitution into planar lipid bilayers. Distinct differences were observed in the calcium-activation and -inactivation properties of the two isoforms. The fish alpha isoform, expressed alone in extraocular muscles, closely resembled the rabbit skeletal muscle RYR. Maximum [3H]ryanodine binding and maximum open probability (Po) of the alpha RYR were achieved from 1 to 10 microM free Ca2+. Millimolar Ca2+ reduced [3H]ryanodine binding and Po close to zero. The beta isoform more closely resembled the fish cardiac RYR in Ca2+ activation of [3H]ryanodine binding. The most prominent difference of the beta and cardiac isoforms from the alpha isoform was the lack of inactivation of [3H]ryanodine binding and Po by millimolar free Ca2+. Differences in activation of [3H]ryanodine binding by adenine nucleotides and inhibition by Mg2+ suggest that the beta and cardiac RYRs are not identical, however. [3H]ryanodine binding by the alpha RYR was selectively inhibited by 100 microM tetracaine, whereas cardiac and beta RYRs were much less affected. Tetracaine can thus be used to separate the properties of the alpha and beta RYRs in preparations in which both are present. The distinct physiological properties of the alpha and beta RYRs that are present together in most vertebrate muscles support models of EC coupling incorporating both directly coupled and Ca(2+)-coupled channels within a single triad junction.
Topics: Animals; Calcium; Calcium Channels; Fishes; Ion Channel Gating; Muscle Proteins; Muscles; Myocardium; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Tetracaine
PubMed: 7696500
DOI: 10.1016/S0006-3495(95)80208-0 -
British Journal of Pharmacology Nov 2022Statins, inhibitors of HMG-CoA reductase, are mainstay treatment for hypercholesterolaemia. However, muscle pain and weakness prevent many patients from benefiting from...
BACKGROUND AND PURPOSE
Statins, inhibitors of HMG-CoA reductase, are mainstay treatment for hypercholesterolaemia. However, muscle pain and weakness prevent many patients from benefiting from their cardioprotective effects. We previously demonstrated that simvastatin activates skeletal ryanodine receptors (RyR1), an effect that could be important in initiating myopathy. Using a range of structurally diverse statin analogues, we examined structural features associated with RyR1 activation, aiming to identify statins lacking this property.
EXPERIMENTAL APPROACH
Compounds were screened for RyR1 activity utilising [ H]ryanodine binding. Mechanistic insight into RyR1 activity was studied by incorporating RyR1 channels from sheep, mouse or rabbit skeletal muscle into bilayers.
KEY RESULTS
All UK-prescribed statins activated RyR1 at nanomolar concentrations. Cerivastatin, withdrawn from the market due to life-threatening muscle-related side effects, was more effective than currently-prescribed statins and possessed the unique ability to open RyR1 channels independently of cytosolic Ca . We synthesised the one essential structural moiety that all statins must possess for HMG-CoA reductase inhibition, the R-3,5-dihydroxypentanoic acid unit, and it did not activate RyR1. We also identified five analogues retaining potent HMG-CoA reductase inhibition that inhibited RyR1 and four that lacked the ability to modulate RyR1.
CONCLUSION AND IMPLICATIONS
That cerivastatin activates RyR1 most strongly supports the hypothesis that RyR1 activation is implicated in statin-induced myopathy. Demonstrating that statin regulation of RyR1 and HMG-CoA reductase are separable effects will allow the role of RyR1 in statin-induced myopathy to be further elucidated by the tool compounds we have identified, allowing development of effective cardioprotective statins with improved patient tolerance.
Topics: Acyl Coenzyme A; Animals; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Mice; Muscle, Skeletal; Muscular Diseases; Rabbits; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sheep; Simvastatin
PubMed: 35703154
DOI: 10.1111/bph.15893 -
The Journal of Biological Chemistry Nov 1996An oligonucleotide probe to a conserved 3' region within the three identified ryanodine receptor-calcium release channel isoforms hybridized to a single clone from a...
An oligonucleotide probe to a conserved 3' region within the three identified ryanodine receptor-calcium release channel isoforms hybridized to a single clone from a rabbit kidney cDNA library. The kidney clone encoded the carboxyl-terminal 338 amino acids within the putative transmembrane domain of the type 2 ryanodine receptor sequence. Reverse transcriptase-polymerase chain reaction with isoform-specific oligonucleotide primers demonstrated the presence of the type 2 ryanodine receptor transcript in rabbit kidney, as well as in a non-excitable cell line, LLC-RK1, derived from rabbit kidney epithelial cells. Amplification by rapid amplification of 5' cDNA ends indicated the kidney type 2 ryanodine receptor transcript extended >7000 base pairs from the stop codon and is therefore not homologous to the short RyR-1 transcript of approximately 2500 base pairs previously observed in rabbit brain. [3H]Ryanodine binding and immunoblot analysis with a type 2 ryanodine receptor-specific antibody demonstrated that the native type 2 ryanodine receptor protein is expressed in the kidney. These observations suggest that the type 2 ryanodine receptor isoform may play a functional role in regulating intracellular calcium homeostasis in non-excitable cells.
Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Western; Brain; Calcium Channels; Cloning, Molecular; DNA, Complementary; Epithelial Cells; Epithelium; Kidney Cortex; Molecular Sequence Data; Muscle Proteins; RNA, Messenger; Rabbits; Radioligand Assay; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sequence Homology, Amino Acid; Tritium
PubMed: 8939887
DOI: 10.1074/jbc.271.47.29583 -
The Journal of Biological Chemistry Oct 1990A high affinity [3H]ryanodine receptor has been solubilized from rabbit brain membranes and biochemically characterized. [3H]Ryanodine binding to rabbit brain membranes...
A high affinity [3H]ryanodine receptor has been solubilized from rabbit brain membranes and biochemically characterized. [3H]Ryanodine binding to rabbit brain membranes is specific and saturable, with a Kd of 1.3 nM. [3H]Ryanodine binding is enriched in membranes from the hippocampus but is significantly lower in membranes from the brain stem and spinal cord. Approximately 60% of [3H]ryanodine-labeled receptor is solubilized from brain membranes using 2.5% CHAPS and 10 mg/ml phosphatidylcholine containing 1 M NaCl. The solubilized brain [3H]ryanodine receptor sediments through sucrose gradients like the skeletal receptor as a large (approximately 30 S) complex. Solubilized receptor is specifically immunoprecipitated by sheep polyclonal antibodies against purified skeletal muscle ryanodine receptor coupled to protein A-Sepharose. [3H]Ryanodine-labeled receptor binds to heparin-agarose, and a protein of approximately 400,000 Da, which is cross-reactive with two polyclonal antibodies raised against the skeletal muscle ryanodine receptor, elutes from the column and is enriched in peak [3H]ryanodine binding fractions. These results suggest that the approximately 400,000-Da protein is the brain form of the high affinity ryanodine receptor and that it shares several properties with the skeletal ryanodine receptor including a large oligomeric structure composed of approximately 400,000-Da subunits.
Topics: Animals; Brain Chemistry; Cell Membrane; Centrifugation, Density Gradient; Chromatography, Affinity; Heparin; Molecular Weight; Precipitin Tests; Rabbits; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Solubility
PubMed: 2211713
DOI: No ID Found -
Proceedings of the National Academy of... Nov 2001In single-channel recordings, the rabbit cardiac Ca(2+) release channel (RyR2) is converted to a fully open subconductance state with about 50% of full conductance by...
In single-channel recordings, the rabbit cardiac Ca(2+) release channel (RyR2) is converted to a fully open subconductance state with about 50% of full conductance by micromolar concentrations of ryanodine. At +30 mV, corresponding to a luminal to cytoplasmic cation current, the probability of opening (P(o)) of ryanodine-modified channels was only marginally altered at pCa 10 (pCa = -log(10) Ca concentration). However, at -30 mV, the P(o) was highly sensitive to Ca(2+) added to the cis (cytoplasmic) side and, at pCa 10, was reduced to less than 0.27. The EC(50) value for channel opening was about pCa 8. No significant Ca(2+) inactivation was observed for ryanodine-modified channels at either -30 mV or +30 mV. The opening of unmodified Ca(2+) channels is Ca(2+) sensitive, with an EC(50) value of about pCa 6 (two orders of magnitude less sensitive than ryanodine-modified channels) and IC(50) values of pCa 2.2 at -30 mV and 2.5 at +30 mV. Mg(2+) decreased the P(o) of ryanodine-modified channels at low Ca(2+) concentrations at both -30 and +30 mV. Caffeine, ATP, and ruthenium red were modulators of the P(o) of ryanodine-modified channels. In a [(3)H]ryanodine binding assay, [(3)H]ryanodine dissociation from the high-affinity binding site was found to be Ca(2+) sensitive, with an IC(50) of pCa 7.1. High concentrations of unlabeled ryanodine prevented [(3)H]ryanodine dissociation, but ruthenium red accelerated dissociation. These results suggest that ryanodine sensitizes Ca(2+) activation of the Ca(2+) release channel and desensitizes Ca(2+) inactivation through an allosteric interaction. [(3)H]Ryanodine dissociates from the high-affinity site when the channel is closed by removal of Ca(2+), implying that high-affinity ryanodine and Ca(2+) binding sites are linked through either short- or long-range interactions, probably involving conformational changes.
Topics: Animals; Calcium; Calcium Channels; Electrophysiology; Ion Channel Gating; Myocardium; Rabbits; Ryanodine; Ryanodine Receptor Calcium Release Channel
PubMed: 11698671
DOI: 10.1073/pnas.241516898 -
Journal of Biochemistry Oct 1992The two ryanodine-binding proteins (RyBPs) have been purified from sarcoplasmic reticulum of bullfrog skeletal muscle by Mono Q column chromatography following...
The two ryanodine-binding proteins (RyBPs) have been purified from sarcoplasmic reticulum of bullfrog skeletal muscle by Mono Q column chromatography following solubilization of SR by CHAPS and sucrose density gradient centrifugation. We conclude that the two RyBPs (alpha- and beta-RyBP) are isoforms on the basis (i) that each RyBP is distinguished by a specific polyclonal antibody and (ii) that distinct polypeptides are generated by limited tryptic digestion of the two RyBPs. Monomeric molecular weights for alpha- and beta-RyBP are estimated to be (690 +/- 10) and (570 +/- 10) kDa, respectively, as determined from mobilities on disc SDS-PAGE using the Weber-Osborn buffer system without 6 M urea, which gives an estimate of (590 +/- 10) kDa for RyBP of rabbit skeletal muscle. Similar determination in the presence of 6 M urea gave 630 kDa for alpha-RyBP and unchanged estimates for the other RyBPs. Both RyBPs show [3H]ryanodine-binding activities which are activated by Ca2+, AMPOPCP, and caffeine, and inhibited by ruthenium red, MgCl2, and procaine. beta-RyBP, however, has higher affinity for Ca2+. In the presence of Ca2+ and AMPOPCP, both RyBPs show single homogeneous binding sites for [3H]ryanodine with Kd = 2-5 nM. The values of Bmax for alpha- and beta-RyBP were 320-340 and 320-375 pmol/mg protein, respectively. These results are consistent with the conclusion that a homo-tetramer of each RyBP binds one ryanodine molecule, taking account of the estimated molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Animals; Calcium; Calcium Channels; Kinetics; Lipid Bilayers; Membranes; Muscles; Protein Binding; Rana catesbeiana; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Tritium
PubMed: 1337084
DOI: 10.1093/oxfordjournals.jbchem.a123931