The Experts below are selected from a list of 29205 Experts worldwide ranked by ideXlab platform
Angela F. Dulhunty - One of the best experts on this subject based on the ideXlab platform.
-
Ubiquitous SPRY domains and their role in the skeletal type Ryanodine Receptor
European Biophysics Journal, 2009Co-Authors: Marco G Casarotto, Angela F. DulhuntyAbstract:We recently identified the second of three SPRY domains in the skeletal muscle Ryanodine Receptor type 1 (RyR1) as a potential binding partner in the RyR1 ion channel for the recombinant II–III loop of the skeletal muscle dihydropyridine Receptor, for a scorpion toxin, Imperatoxin A and for an interdomain interaction within RyR1. SPRY domains are structural domains that were first described in the fungal Dictyostelium discoideum tyrosine kinase spore lysis A and all three isoforms of the mammalian Ryanodine Receptor (RyR). Our studies are the first to assign a function to any of the three SPRY domains in the RyR. However, in other systems SPRY domains provide binding sites for regulatory proteins or intramolecular binding sites that maintain the structural integrity of a protein. In this article, we review the general characteristics of a range of SPRY domains and discuss evidence that the SPRY2 domain in RyR1 supports interactions with binding partners that contain a structural surface of aligned basic residues.
-
Effects of an α-helical Ryanodine Receptor C-terminal tail peptide on Ryanodine Receptor activity: Modulation by Homer
The International Journal of Biochemistry & Cell Biology, 2006Co-Authors: Pierre Pouliquin, Marco G Casarotto, Francesco Zorzato, Esther M. Gallant, Suzy M. Pace, Suzanne M. Curtis, Peta J. Harvey, Angela F. DulhuntyAbstract:Abstract We have determined the structure of a domain peptide corresponding to the extreme 19 C-terminal residues of the Ryanodine Receptor Ca2+ release channel. We examined functional interactions between the peptide and the channel, in the absence and in the presence of the regulatory protein Homer. The peptide was partly α-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K+ channels. The peptide (0.1–10 μM) inhibited skeletal Ryanodine Receptor channels when the cytoplasmic Ca2+ concentration was 1 μM; but with 10 μM cytoplasmic Ca2+, skeletal Ryanodine Receptors were activated by ≤1.0 μM peptide and inhibited by 10 μM peptide. Cardiac Ryanodine Receptors on the other hand were inhibited by all peptide concentrations, at both Ca2+ concentrations. When channels did open in the presence of the peptide, they were more likely to open to substate levels. The inhibition and increased fraction of openings to subconductance levels suggested that the domain peptide might destabilise inter-domain interactions that involve the C-terminal tail. We found that Homer 1b not only interacts with the channels, but reduces the inhibitory action of the C-terminal tail peptide, perhaps by stabilizing inter-domain interactions and preventing their disruption.
-
Calsequestrin is an inhibitor of skeletal muscle Ryanodine Receptor calcium release channels.
Biophysical journal, 2002Co-Authors: Nicole A. Beard, Angela F. Dulhunty, Magdalena M Sakowska, Derek R. LaverAbstract:We provide novel evidence that the sarcoplasmic reticulum calcium binding protein, calsequestrin, inhibits native Ryanodine Receptor calcium release channel activity. Calsequestrin dissociation from junctional face membrane was achieved by increasing luminal (trans) ionic strength from 250 to 500 mM with CsCl or by exposing the luminal side of Ryanodine Receptors to high [Ca(2+)] (13 mM) and dissociation was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Calsequestrin dissociation caused a 10-fold increase in the duration of Ryanodine Receptor channel opening in lipid bilayers. Adding calsequestrin back to the luminal side of the channel after dissociation reversed this increased activity. In addition, an anticalsequestrin antibody added to the luminal solution reduced Ryanodine Receptor activity before, but not after, calsequestrin dissociation. A population of Ryanodine Receptors (approximately 35%) may have initially lacked calsequestrin, because their activity was high and was unaffected by increasing ionic strength or by anticalsequestrin antibody: their activity fell when purified calsequestrin was added and they then responded to antibody. In contrast to native Ryanodine Receptors, purified channels, depleted of triadin and calsequestrin, were not inhibited by calsequestrin. We suggest that calsequestrin reduces Ryanodine Receptor activity by binding to a coprotein, possibly to the luminal domain of triadin.
-
actions of sulfhydryl reagents on single Ryanodine Receptor ca 2 release channels from sheep myocardium
American Journal of Physiology-cell Physiology, 1997Co-Authors: K R Eager, L D Roden, Angela F. DulhuntyAbstract:Effects of the reactive disulfides, 2,2'- and 4,4'-dithiodipyridine, on single cardiac Ryanodine Receptor (RyR) ion channels incorporated into lipid bilayers are reported. RyRs are activated within...
-
single channel activity of the Ryanodine Receptor calcium release channel is modulated by fk 506
FEBS Letters, 1994Co-Authors: Gerard P Ahern, Pauline R Junankar, Angela F. DulhuntyAbstract:The immunosuppressant drug FK-506 (3-20 microM) increased the open probability of Ryanodine Receptor calcium release channels, formed by incorporation of terminal cisternae vesicles from rabbit skeletal muscle into lipid bilayers, with cis (cytoplasmic) calcium concentrations between 10(-7) M and 10(-3) M. FK-506 increased mean current and channel open time and induced long sojourns at subconductance levels that were between 28% and 38% of the maximum conductance and were distinct from the Ryanodine-induced subconductance level at about 45% of the maximum conductance. FK-506 relieved the Ca2+ inactivation of the Ryanodine Receptor seen at 10(-3) M Ca2+. The results are consistent with FK-506 removal of FK-506 binding protein from the Ryanodine Receptor.
Martin Hohenegger - One of the best experts on this subject based on the ideXlab platform.
-
Use‐dependent inhibition of the skeletal muscle Ryanodine Receptor by the suramin analogue NF676
British Journal of Pharmacology, 2005Co-Authors: Ilse Wolner, Matthias U. Kassack, Heiko Ullmann, Anton Karel, Martin HoheneggerAbstract:The skeletal muscle Ca2+ release channel, the Ryanodine Receptor, is activated by the trypanocidal drug suramin via the calmodulin-binding site. As calmodulin activates and inhibits the Ryanodine Receptor depending on whether Ca2+ is absent or present, suramin analogues were screened for inhibition of the Ryanodine Receptor. Up to 300 μM, the novel suramin analogue, 4,4′-(carbonyl-bis(imino-4,1-phenylene-(2,5-benzimidazolylene)carbonylimino))-bis-benzenesulfonic acid disodium salt (NF676) was not able to significantly inhibit the basal [3H]Ryanodine binding. However, kinetic analysis of the high affinity [3H]Ryanodine binding elucidates a time-dependent increment of inhibition by NF676, which is indicative for an open channel blocker. Moreover, the Ryanodine Receptor was much more sensitive towards inhibition by NF676 when preactivated with caffeine or the nonhydrolysable ATP analogue, adenylyl-imidodiphosphate. Nonetheless, the suramin activated Ryanodine Receptor was not susceptible towards high-affinity NF676 inhibition, indicating an allosteric hindrance between the binding sites of suramin and NF676. In the line of this finding, NF676 per se was not capable to elute the purified Ryanodine Receptor from a calmodulin-Sepharose, but it prevented the elution by suramin. Other than suramin, NF676 did not inhibit the Ca2+ ATPase of the sarcoplasmic reticulum. However, suramin-induced Ca2+ release from sarcoplasmic reticulum was completely abrogated by preincubation with NF676. Taken together, we conclude from these data that NF676 represents a novel lead compound as a potent use-dependent blocker of the skeletal muscle Ryanodine Receptor via an allosteric interaction with the suramin-binding site. British Journal of Pharmacology (2005) 146, 525–533. doi:10.1038/sj.bjp.0706359
-
Pharmacological activation of the Ryanodine Receptor in Jurkat T-lymphocytes.
British Journal of Pharmacology, 1999Co-Authors: Martin Hohenegger, Ingeborg Berg, Lukas Weigl, Georg W. Mayr, Barry V. L. Potter, Andreas H. GuseAbstract:Recently, we provided evidence for cyclic adenosine 5′-diphosphate-ribose, cADP-ribose, as a second messenger in Jurkat T-lymphocytes upon stimulation of the T-cell Receptor/CD3- complex (Guse et al., 1999). cADP-ribose mobilizes Ca2+ from an intracellular Ca2+ store which is sensitive to caffeine and gated by the Ryanodine Receptor/Ca2+ release channel. In the present study we investigated the ability of the trypanocidal drug, suramin, to activate the Ryanodine Receptor of T-cells. Since suramin cannot permeate the plasma membrane, it was necessary to microinject the drug into Fura-2 loaded T-lymphocytes. In a dose dependent manner suramin increased the intracellular Ca2+ concentration. The dose-response curve is very steep and calculates for an EC50 of 7.6±2.9 mM suramin in the injection pipette. Co-injection of the selective Ryanodine Receptor inhibitor ruthenium red completely abolished the suramin induced Ca2+ transient. This finding allows for the conclusion that the IP3-Receptor sensitive Ca2+ pool is not the primary target of the suramin induced Ca2+ transient. Furthermore, Ins(1,4,6)PS3, an antagonist of the InsP3-Receptor could not suppress the suramin-induced Ca2+ signal. The suramin induced Ca2+ transients declined very slowly; however, in the presence of Ins(1,4,6)PS3 this decay was accelerated. In addition, suramin did not interact with the cADP-ribose binding site of the Ryanodine Receptor of T-cells. In conclusion, suramin is found to be an agonist for the T-cell Ryanodine Receptor as previously found for the cardiac and skeletal muscle isoform. Therefore, suramin can be designated a universal Ryanodine Receptor agonist. British Journal of Pharmacology (1999) 128, 1235–1240; doi:10.1038/sj.bjp.0702935
-
Suramin and Suramin Analogs Activate Skeletal Muscle Ryanodine Receptor via a Calmodulin Binding Site
Molecular Pharmacology, 1999Co-Authors: Markus Klinger, Michael Freissmuth, Peter Nickel, Margit Stäbler-schwarzbart, Matthias U. Kassack, Josef Suko, Martin HoheneggerAbstract:Contraction of skeletal muscle is triggered by the rapid release of Ca2+ from the sarcoplasmic reticulum via the Ryanodine Receptor/calcium-release channel. The trypanocidal drug suramin is an efficient activator of the Ryanodine Receptor. Here, we used high-affinity [3H]Ryanodine binding to sarcoplasmic reticulum from rabbit skeletal muscle to screen for more potent analogs of suramin. This approach resulted in the identification of NF307, which accelerates the association rate of [3H]Ryanodine binding with an EC50 = 91 ± 7 μM at 0.19 μM calculated free Ca2+. In single-channel recordings with the purified Ryanodine Receptor, NF307 increased mean open probability at 0.6 μM Ca2+ from 0.020 ± 0.006 to 0.53 ± 0.07 with no effect on current amplitude and unitary conductance. Like caffeine, NF307 exerts a very pronounced Ca2+-sensitizing effect (EC50 of Ca2+ shifted ∼10-fold by saturating NF307 concentrations). Conversely, increasing concentrations of free Ca2+ sensitized the Receptor for NF307 (EC50 = 14.6 ± 3.5 μM at 0.82 μM estimated free Ca2+). The effects of NF307 and caffeine on [3H]Ryanodine binding were additive, irrespective of the Ca2+concentration. In contrast, the effects of calmodulin, which activates and inhibits the Ryanodine Receptor in the absence and presence of Ca2+, respectively, and of NF307 were mutually antagonistic. If the purified Ryanodine Receptor was prebound to a calmodulin-Sepharose matrix, 100 μM NF307 and 300 μM suramin eluted the purified Ryanodine Receptor to an extent that was comparable to the effect of 10 μM calmodulin. We conclude that NF307 and suramin interact directly with a calmodulin binding domain of the Ryanodine Receptor. Because of its potent calcium-sensitizing effect, NF307 may represent a lead compound in the search of synthetic Ryanodine Receptor ligands.
-
Activation of the Skeletal Muscle Ryanodine Receptor by Suramin and Suramin Analogs
Molecular Pharmacology, 1996Co-Authors: Martin Hohenegger, Marina Matyash, Kati Poussu, Annegret Herrmann-frank, Sándor Sárközi, Frank Lehmann-horn, Michael FreissmuthAbstract:SUMMARY Ca2� release from skeletal muscle sarcoplasmic reticulum is activated by adenine nucleotides and suramin. Because suramin is known to interact with ATP-binding enzymes and ATP Receptors (P2-purinergic Receptors), the stimulation by suramin has been postulated to occur via the adenine nudeotide-binding site of the Ryanodine Receptor/Ca2�-release channel. We tested this hypothesis using suramin and the following suramin analogs: NF037, NFO18, NF023, and NFOO7. The suramin analogs stimulate the binding of [�H]Ryanodine binding to sarcoplasmic reticulum membranes with the following rank order of potency: suramin (EC50 = -60 �tM) > NF037 (EC50 = .- 150 p.M) > NFO18 > NF023 > NFOO7. The suramininduced stimulation occurs via a myoplasmic binding site on the Ryanodine Receptor as confirmed by binding experiments and single-channel recordings with the purified protein. This
-
Activation and labelling of the purified skeletal muscle Ryanodine Receptor by an oxidized ATP analogue.
Biochemical Journal, 1995Co-Authors: Martin Hohenegger, Annegret Herrmann-frank, Michael Richter, Frank Lehmann-hornAbstract:: 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.
Francesco Zorzato - One of the best experts on this subject based on the ideXlab platform.
-
Effects of an α-helical Ryanodine Receptor C-terminal tail peptide on Ryanodine Receptor activity: Modulation by Homer
The International Journal of Biochemistry & Cell Biology, 2006Co-Authors: Pierre Pouliquin, Marco G Casarotto, Francesco Zorzato, Esther M. Gallant, Suzy M. Pace, Suzanne M. Curtis, Peta J. Harvey, Angela F. DulhuntyAbstract:Abstract We have determined the structure of a domain peptide corresponding to the extreme 19 C-terminal residues of the Ryanodine Receptor Ca2+ release channel. We examined functional interactions between the peptide and the channel, in the absence and in the presence of the regulatory protein Homer. The peptide was partly α-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K+ channels. The peptide (0.1–10 μM) inhibited skeletal Ryanodine Receptor channels when the cytoplasmic Ca2+ concentration was 1 μM; but with 10 μM cytoplasmic Ca2+, skeletal Ryanodine Receptors were activated by ≤1.0 μM peptide and inhibited by 10 μM peptide. Cardiac Ryanodine Receptors on the other hand were inhibited by all peptide concentrations, at both Ca2+ concentrations. When channels did open in the presence of the peptide, they were more likely to open to substate levels. The inhibition and increased fraction of openings to subconductance levels suggested that the domain peptide might destabilise inter-domain interactions that involve the C-terminal tail. We found that Homer 1b not only interacts with the channels, but reduces the inhibitory action of the C-terminal tail peptide, perhaps by stabilizing inter-domain interactions and preventing their disruption.
-
Ryanodine Receptor 1 mutations, dysregulation of calcium homeostasis and neuromuscular disorders.
Neuromuscular disorders : NMD, 2005Co-Authors: Susan Treves, Ayuk A Anderson, Sylvie Ducreux, Alexandra Divet, Christophe Bleunven, Cristiano Grasso, Silvia Paesante, Francesco ZorzatoAbstract:The skeletal muscle Ryanodine Receptor is an intracellular calcium release channel which plays a central role in excitation contraction coupling. At least 80 mutations have been identified in the gene encoding the skeletal muscle Ryanodine Receptor and linked to several neuromuscular disorders, whose common feature appears to be a dysregulation of calcium homeostasis. A decade of research into the functional consequences of how these mutations affect the functional properties of the Ryanodine Receptor and their impact on disease, have significantly advanced our understanding of Malignant Hyperthermia, Central Core Disease and Multiminicore Disease. This review gives an overview of the important findings in the field of calcium homeostasis in skeletal muscle and describes how mutations in the Ryanodine Receptor gene might affect the function of this intracellular calcium release channel and lead to neuromuscular disorders.
-
Chlorocresol: an activator of Ryanodine Receptor-mediated Ca2+ release.
Molecular Pharmacology, 1993Co-Authors: Francesco Zorzato, Erica Scutari, Vincenzo Tegazzin, E Clementi, Susan TrevesAbstract:: In the present study we investigated the effect of the compound chlorocresol on intracellular Ca2+ homeostasis. Three different systems that have been shown to express the Ryanodine Receptor Ca2+ channel were chosen, i.e., skeletal muscle sarcoplasmic reticulum, cerebellar microsomes, and PC12 cells. In skeletal muscle sarcoplasmic reticulum, 4-chloro-m-cresol was found to be a potent activator of Ca2+ release mediated by a ruthenium red/caffeine-sensitive Ca2+ release channel. In cerebellar microsomes, this compound released Ca2+ from an inositol-1,4,5-trisphosphate-insensitive store, suggesting that there too it was acting at the Ryanodine Receptor level. When tested on PC12 cells, chlorocresol released Ca2+ from a caffeine- and thapsigargin-sensitive intracellular store. In addition, the compound was capable of releasing Ca2+ after pretreatment of PC12 cells with bradykinin, suggesting that it acts on a channel contained within an intracellular Ca2+ store that is distinct from that sensitive to inositol-1,4,5-trisphosphate. Structure-activity relationship analyses suggest that the chloro and methyl groups in chlorocresols are important for the activation of the Ryanodine Receptor Ca2+ release channel.
K. Jane Grande-allen - One of the best experts on this subject based on the ideXlab platform.
-
The Ryanodine Receptor Contributes to the Lysophosphatidylcholine-Induced Mineralization in Valvular Interstitial Cells
Cardiovascular Engineering and Technology, 2020Co-Authors: Reid L. Wilson, Christopher B. Sylvester, Dena C. Wiltz, Aditya Kumar, Tahir H. Malik, Joel D. Morrisett, K. Jane Grande-allenAbstract:Purpose Fibrocalcific aortic valve disease (CAVD) is caused by the deposition of calcific nodules in the aortic valve leaflets, resulting in progressive loss of function that ultimately requires surgical intervention. This process is actively mediated by the resident valvular interstitial cells (VICs), which, in response to oxidized lipids, transition from a quiescent to an osteoblast-like state. The purpose of this study was to examine if the Ryanodine Receptor, an intracellular calcium channel, could be therapeutically targeted to prevent this phenotypic conversion. Methods The expression of the Ryanodine Receptor in porcine aortic VICs was characterized by qRT-PCR and immunofluorescence. Next, the VICs were exposed to lysophosphatidylcholine, an oxidized lipid commonly found in low-density lipoprotein, while the activity of the Ryanodine Receptor was modulated with Ryanodine. The cultures were analyzed for markers of cellular mineralization, alkaline phosphatase activity, proliferation, and apoptosis. Results Porcine aortic VICs predominantly express isoform 3 of the Ryanodine Receptors, and this protein mediates the cellular response to LPC. Exposure to LPC caused elevated intracellular calcium concentration in VICs, raised levels of alkaline phosphatase activity, and increased calcific nodule formation, but these changes were reversed when the activity of the Ryanodine Receptor was blocked. Conclusions Our findings suggest blocking the activity of the Ryanodine Receptor can attenuate the valvular mineralization caused by LPC. We conclude that oxidized lipids, such as LPC, play an important role in the development and progression of CAVD and that the Ryanodine Receptor is a promising target for pharmacological intervention.
-
The Ryanodine Receptor Contributes to the Lysophosphatidylcholine-Induced Mineralization in Valvular Interstitial Cells.
Cardiovascular engineering and technology, 2020Co-Authors: Reid L. Wilson, Christopher B. Sylvester, Dena C. Wiltz, Aditya Kumar, Tahir H. Malik, Joel D. Morrisett, K. Jane Grande-allenAbstract:PURPOSE Fibrocalcific aortic valve disease (CAVD) is caused by the deposition of calcific nodules in the aortic valve leaflets, resulting in progressive loss of function that ultimately requires surgical intervention. This process is actively mediated by the resident valvular interstitial cells (VICs), which, in response to oxidized lipids, transition from a quiescent to an osteoblast-like state. The purpose of this study was to examine if the Ryanodine Receptor, an intracellular calcium channel, could be therapeutically targeted to prevent this phenotypic conversion. METHODS The expression of the Ryanodine Receptor in porcine aortic VICs was characterized by qRT-PCR and immunofluorescence. Next, the VICs were exposed to lysophosphatidylcholine, an oxidized lipid commonly found in low-density lipoprotein, while the activity of the Ryanodine Receptor was modulated with Ryanodine. The cultures were analyzed for markers of cellular mineralization, alkaline phosphatase activity, proliferation, and apoptosis. RESULTS Porcine aortic VICs predominantly express isoform 3 of the Ryanodine Receptors, and this protein mediates the cellular response to LPC. Exposure to LPC caused elevated intracellular calcium concentration in VICs, raised levels of alkaline phosphatase activity, and increased calcific nodule formation, but these changes were reversed when the activity of the Ryanodine Receptor was blocked. CONCLUSIONS Our findings suggest blocking the activity of the Ryanodine Receptor can attenuate the valvular mineralization caused by LPC. We conclude that oxidized lipids, such as LPC, play an important role in the development and progression of CAVD and that the Ryanodine Receptor is a promising target for pharmacological intervention.
Andrew R. Marks - One of the best experts on this subject based on the ideXlab platform.
-
Ryanodine Receptor dysfunction in human disorders
Biochimica et Biophysica Acta, 2018Co-Authors: Alexander Kushnir, Benjamin Wajsberg, Andrew R. MarksAbstract:Regulation of intracellular calcium (Ca2+) is critical in all cell types. The Ryanodine Receptor (RyR), an intracellular Ca2+ release channel located on the sarco/endoplasmic reticulum (SR/ER), releases Ca2+ from intracellular stores to activate critical functions including muscle contraction and neurotransmitter release. Dysfunctional RyR-mediated Ca2+ handling has been implicated in the pathogenesis of inherited and non-inherited conditions including heart failure, cardiac arrhythmias, skeletal myopathies, diabetes, and neurodegenerative diseases. Here we have reviewed the evidence linking human disorders to RyR dysfunction and describe novel approaches to RyR-targeted therapeutics.
-
ca2 calmodulin dependent protein kinase ii phosphorylation regulates the cardiac Ryanodine Receptor
Circulation Research, 2004Co-Authors: Xander H T Wehrens, Stephan E Lehnart, Steven Reiken, Andrew R. MarksAbstract:The cardiac Ryanodine Receptor (RyR2)/calcium release channel on the sarcoplasmic reticulum is required for muscle excitation-contraction coupling. Using site-directed mutagenesis, we identified th...
-
Single channel properties and calcium conductance of the cloned expressed Ryanodine Receptor/calcium-release channel.
Society of General Physiologists series, 1996Co-Authors: Karol Ondrias, Scott A, Barbara E. Ehrlich, Andrew R. MarksAbstract:The calcium-release channel/Ryanodine Receptor of the sarcoplasmic reticulum is a 2.3 million-D structure required for intracellular calcium release during excitation-contraction coupling in skeletal muscle. This structure is the largest ion channel characterized to date and is composed of four 565,000-D Ryanodine Receptors plus four molecules of FKBP12. In the present study we describe the single channel properties of the cloned expressed Ryanodine Receptor, with and without FKBP12, reconstituted into planar lipid bilayers with Ca as the charge carrier. The conductance for Ca (luminal, 53 mM/cytoplasmic, 10 microM) was 103 pS for the cloned expressed RyR and for the native channel from rabbit skeletal muscle. Conductance through the channel was Ca dependent: A decrease in the Ca gradient to luminal 10.6/cytoplasmic 10 microM reduced conductance to 68 pS for both the cloned and native RyR. The recombinant Ryanodine Receptor consistently behaved like the native skeletal muscle channel in terms of activation by caffeine, calcium, and ATP; inhibition by ruthenium red; and modulation by Ryanodine. In the absence of FKBP12, the cloned expressed RyR exhibited multiple subconductance states and addition of FKBP12 reduced the frequency of subconductance states. These results show that with Ca as the charge carrier, the single channel properties of the cloned expressed RyR plus FKBP12 are essentially the same as those of the native channel.
-
Reduced Ryanodine Receptor Content in Isolated Neonatal Cardiomyocytes Compared with the Intact Tissue
Journal of Molecular and Cellular Cardiology, 1994Co-Authors: Melinda Fitzgerald, Andrew R. Marks, Craig B. Neylon, Elizabeth A. WoodcockAbstract:Abstract The effects of isolation and culture of rat neonatal ventricular myocytes on the properties of the Ryanodine Receptor were investigated. [ 3 H]-Ryanodine bound to a single class of sites in membranes prepared from intact neonatal ventricle, with an affinity of 16.3 ± 2.8 nM (mean ± S.E.; n = 3) and a capacity of 546 ± 64 fmol/mg protein (mean ±S.E.; n = 3). In contrast, no detectable displaceable binding of [ 3 H]-Ryanodine was observed when similar experiments were performed using membranes prepared from isolated neonatal cardiomyocytes. The apparent absence of [ 3 H]-Ryanodine binding in the neonatal cardiomyocytes suggested either reduced Ryanodine Receptor protein or the conversion of the Receptors to a low affinity state. To distinguish between these possibilities, the content of Ryanodine Receptor protein was measured using SDS-PAGE followed by western blotting. Membranes prepared from neonatal ventricle contained substantial amounts of Ryanodine Receptors, as demonstrated by a dense band on western blots. However the corresponding band in preparations of isolated cells, while having similar electrophoretic mobility, was barely detectable. It is concluded that the Ryanodine Receptor protein is strongly expressed in intact neonatal ventricle, but the level of expression is markedly reduced upon isolation of the cardiomyocytes. These findings demonstrate that Ryanodine Receptors expression is significantly down-regulated when rat neonatal ventricular myocytes are isolated and maintained in culture. This marked decrease in expression of one of the key protein required for excitation-contraction coupling suggests that regulatory factors present in intact myocardium play an important role in maintaining expression of elements of the contractile mechanism.
