The Experts below are selected from a list of 17154 Experts worldwide ranked by ideXlab platform
Angela F. Dulhunty - One of the best experts on this subject based on the ideXlab platform.
-
the inhibitory glutathione transferase m2 2 binding site is located in divergent region 3 of the cardiac ryanodine receptor
Biochemical Pharmacology, 2012Co-Authors: Ruwani Punyakanthi Hewawasam, Yamuna Karunasekara, Marco G Casarotto, Angela F. Dulhunty, Philip G. BoardAbstract:Abstract The muscle-specific glutathione transferase GSTM2-2 modulates the activity of ryanodine receptor (RyR) calcium release channels: it inhibits the activity of cardiac RyR (RyR2) channels with high affinity and activates skeletal RyR (RYR1) channels with low affinity. The C terminal domain of GSTM2-2 (GSTM2C) alone physically binds to RyR2 and inhibits its activity, but it does not bind to RYR1. We have now used yeast two-hybrid analysis, chemical cross-linking, intrinsic tryptophan fluorescence and Ca2+ release studies to determine that the binding site for GSTM2C is in divergent region 3 (D3) of RyR2. The D3 region encompasses residues 1855–1890 in RyR2. Specific mutagenesis shows the binding primarily involves electrostatic interactions with residues K1875, K1886, R1887 and K1889, all residues that are present in RyR2, but not in RYR1. The significant sequence differences between the D3 regions of RyR2 and RYR1 explain why GSTM2-2 specifically inhibits RyR2. This specific inhibition of RyR2 could modulate Ca cycling and be useful for the treatment of heart failure. RyR2 inhibition during diastole may improve filling of the SR with Ca2+ and improve contractility.
-
The elusive role of the SPRY2 domain in RYR1
Channels (Austin Tex.), 2011Co-Authors: Hanshen Tae, Marco G Casarotto, Philip G. Board, Robert T. Dirksen, Lan Wei, Esther M. Gallant, Hermia Willemse, Shamaruh Mirza, Angela F. DulhuntyAbstract:The second of three SPRY domains (SPRY2, S1085 -V1208) located in the skeletal muscle ryanodine receptor (RYR1) is contained within regions of RYR1 that influence EC coupling and bind to imperatoxin A, a toxin probe of RYR1 channel gating. We examined the binding of the F loop (P1107-A1121) in SPRY2 to the ASI/basic region in RYR1 (T3471-G3500, containing both alternatively spliced (ASI) residues and neighboring basic amino acids). We then investigated the possible influence of this interaction on excitation contraction (EC) coupling. A peptide with the F loop sequence and an antibody to the SPRY2 domain each enhanced RYR1 activity at low concentrations and inhibited at higher concentrations. A peptide containing the ASI/basic sequence bound to SPRY2 and binding decreased ~10-fold following mutation or structural disruption of the basic residues. Binding was abolished by mutation of three critical acidic F loop residues. Together these results suggest that the ASI/basic and SPRY2 domains interact in an F loop regulatory module. Although a region that includes the SPRY2 domain influences EC coupling, as does the ASI/basic region, Ca2+ release during ligand- and depolarization-induced RYR1 activation were not altered by mutation of the three critical F loop residues following expression of mutant RYR1 in RYR1-null myotubes. Therefore the electrostatic regulatory interaction between the SPRY2 F loop residues (that bind to imperatoxin A) and the ASI/basic residues of RYR1 does not influence bi-directional DHPR-RYR1 signaling during skeletal EC coupling, possibly because the interaction is interrupted by the influence of factors present in intact muscle cells.
-
3d mapping of the spry2 domain of RYR1 by antibody labeling and single particle cryo em
Biophysical Journal, 2011Co-Authors: Alex Peralvarezmarin, Hanshen Tae, Marco G Casarotto, Philip G. Board, Angela F. Dulhunty, Montserrat SamsoAbstract:The ryanodine receptor (RYR1) and the dihydropyridine receptor (DHPR) interact functionally at the T tubule/sarcoplasmic reticulum interface of skeletal muscle and are main participants of the excitation-contraction coupling process. The SPRY2 domain of RYR1 has been shown to be relevant for the RYR1/ DHPR interaction. Here, using a combination of immunolabeling and single-particle cryo-EM we have mapped SPRY2 in the 3D structure of RYR1. RYR1 was incubated with three different antibodies against the SPRY2 domain and vitrified for cryo-EM imaging. The two main obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the several possible binding locations of the antibody to the tetrameric RYR1, were overcome by modification of the 3D reconstruction scheme. This enabled us to obtain a 3D reconstruction of RYR1 with the antibody bound, which in turn has allowed us to map the SPRY2 domain in a T-tubule facing domain of RYR1.
-
Alternative splicing of RYR1 alters the efficacy of skeletal EC coupling.
Cell calcium, 2009Co-Authors: Takashi Kimura, Marco G Casarotto, Noriaki Ikemoto, Robert T. Dirksen, John D. Lueck, Peta J. Harvey, Suzy M. Pace, Angela F. DulhuntyAbstract:Alternative splicing of ASI residues (Ala(3481)-GIn(3485)) in the skeletal muscle ryanodine receptor (RYR1) is developmentally regulated: the residues are present in adult ASI(+)RYR1, but absent in the juvenile ASI(-)RYR1 which is over-expressed in adult myotonic dystrophy type 1 (DM1). Although this splicing switch may influence RYR1 function in developing muscle and DM1, little is known about the properties of the splice variants. We examined excitation-contraction (EC) coupling and the structure and interactions of the ASI domain (Thr(3471)-Gly(3500)) in the splice variants. Depolarisation-dependent Ca(2+) release was enhanced by >50% in myotubes expressing ASI(-)RYR1 compared with ASI(+)RYR1, although DHPR L-type currents and SIR Ca(2+) content were unaltered, while ASI(-)RYR1 channel function was actually depressed. The effect on EC coupling did not depend on changes in ASI domain secondary structure. Probing RYR1 function with peptides possessing the ASI domain sequence indicated that the domain contributes to an inhibitory module in RYR1. The action of the peptide depended on a sequence of basic residues and their alignment in an alpha-helix adjacent to the ASI splice site. This is the first evidence that the ASI residues contribute to an inhibitory module in RYR1 that influences EC coupling. Implications for development and DM1 are discussed. (c) 2008 Elsevier Ltd. All rights reserved.
-
Independent Actions Of Junctin And Triadin On Skeletal Muscle RYR1 Channels
Biophysical Journal, 2009Co-Authors: Lan Wei, Esther M. Gallant, Nicole A. Beard, Angela F. DulhuntyAbstract:Two similar proteins, junctin and triadin, are associated with the membrane of the sarcoplasmic reticulum (SR) Ca2+ store in skeletal muscle and have been thought to serve a common function in anchoring the Ca2+ binding protein calsequestrin (CSQ1) to the type 1 ryanodine receptor (RYR1). The resulting CSQ1/junctin/triadin/RYR1 complex is thought to mediate a functional response in the ion channel which conserves the Ca2+ stored within the SR. The individual actions of triadin and junctin on RYR1 and their contribution to the functional interaction between CSQ1 and RYR1 have not previously been examined and are reported here. Highly purified triadin or junctin added to the luminal side of purified RYR1 channels in lipid bilayers caused an increase in channel open time and stabilized channel openings to the maximum conductance. Competition studies indicated that triadin and junctin exert independent actions on RYR1. In addition, purified CSQ1 inhibited junctin/triadin-associated or junctin-associated, but not triadin-associated, RYR1 channels in the presence of 1mM luminal Ca2+. As with native RYR1, purified RYR1 channels associated with either CSQ1/triadin/junctin, or CSQ1/junctin were further inhibited when luminal [Ca2+] was reduced from 1mM to ≤100μM, in the same way as the native RYR1 channel complex. In marked contrast, the channel activity of the CSQ1/triadin/RYR1 complex increased when luminal Ca2+ was lowered, in a similar manner to the un-associated purified RYR1. These results indicate that junctin alone is responsible for mediating signals between luminal Ca2+, CSQ1 and RYR1 and that triadin does not contribute to this function. Other evidence indicates that triadin plays an independent role in supporting Ca2+ release during excitation-contraction coupling (Goonasekara et al. 2007 J. gen. Physiol. 130, 365; Wang et al. 2008 Cell Calcium in press).
Paul D. Allen - One of the best experts on this subject based on the ideXlab platform.
-
RYR1 mediated ca2 leak and ca2 entry determine resting intracellular ca2 in skeletal myotubes
Journal of Biological Chemistry, 2010Co-Authors: Jose M Eltit, Paul D. Allen, Isaac N. Pessah, Tianzhong Yang, Tadeusz F Molinski, Jose R LopezAbstract:The control of resting free Ca(2+) in skeletal muscle is thought to be a balance of channels, pumps, and exchangers in both the sarcolemma and sarcoplasmic reticulum. We explored these mechanisms using pharmacologic and molecular perturbations of genetically engineered (dyspedic) muscle cells that constitutively lack expression of the skeletal muscle sarcoplasmic reticulum Ca(2+) release channels, RYR1 and RyR3. We demonstrate here that expression of RYR1 is responsible for more than half of total resting Ca(2+) concentration ([Ca(2+)](rest)) measured in wild type cells. The elevated [Ca(2+)](rest) in RYR1-expressing cells is not a result of active gating of the RYR1 channel but instead is accounted for by the RYR1 ryanodine-insensitive Ca(2+) leak conformation. In addition, we demonstrate that basal sarcolemmal Ca(2+) influx is also governed by RYR1 expression and contributes in the regulation of [Ca(2+)](rest) in skeletal myotubes.
-
RYR1-specific requirement for depolarization-induced Ca2+ sparks in urinary bladder smooth muscle
Journal of Cell Science, 2007Co-Authors: Nicolas Fritz, Jean Luc Morel, Loice H. Jeyakumar, Sidney Fleischer, Paul D. Allen, Jean Mironneau, Nathalie MacrezAbstract:Ryanodine receptor subtype 1 (RYR1) has been primarily characterized in skeletal muscle but several studies have revealed its expression in smooth muscle. Here, we used RYR1 -null mice to investigate the role of this isoform in Ca 2+ signaling in urinary bladder smooth muscle. We show that RYR1 is required for depolarization-induced Ca 2+ sparks, whereas RyR2 and RyR3 are sufficient for spontaneous or caffeine-induced Ca 2+ sparks. Immunostaining revealed specific subcellular localization of RYR1 in the superficial sarcoplasmic reticulum; by contrast, RyR2 and RyR3 are mainly expressed in the deep sarcoplasmic reticulum. Paradoxically, lack of depolarization-induced Ca 2+ sparks in RYR1 –/– myocytes was accompanied by an increased number of cells displaying spontaneous or depolarization-induced Ca 2+ waves. Investigation of protein expression showed that FK506-binding protein (FKBP) 12 and FKBP12.6 (both of which are RyR-associated proteins) are downregulated in RYR1 –/– myocytes, whereas expression of RyR2 and RyR3 are unchanged. Moreover, treatment with rapamycin, which uncouples FKBPs from RyR, led to an increase of RyR-dependent Ca 2+ signaling in wild-type urinary bladder myocytes but not in RYR1 –/– myocytes. In conclusion, although decreased amounts of FKBP increase Ca 2+ signals in RYR1 –/– urinary bladder myocytes the depolarization-induced Ca 2+ sparks are specifically lost, demonstrating that RYR1 is required for depolarization-induced Ca 2+ sparks and suggesting that the intracellular localization of RYR1 fine-tunes Ca 2+ signals in smooth muscle.
-
Occurrence of atypical Ca2+ transients in triadin-binding deficient-RYR1 mutants.
Biochemical and biophysical research communications, 2006Co-Authors: Eun Hui Lee, Gerhard Meissner, Paul D. Allen, Dong Woo Song, Jae Man Lee, Han KimAbstract:Triadin in the junctional sarcoplasmic reticulum (SR) of skeletal muscle cells has been suggested to interact with ryanodine receptor 1 (RYR1) via its KEKE motifs. Recently, we showed that amino acid residues D4878, D4907, and E4908 in RYR1 are critical for triadin-binding in vitro [J.M. Lee, S.H. Rho, D.W. Shin, C. Cho, W.J. Park, S.H. Eom, J. Ma, D.H. Kim, Negatively charged amino acids within the intraluminal loop of ryanodine receptor are involved in the interaction with triadin, J. Biol. Chem. 279 (2004) 6994–7000]. In order to test whether a disruption of the triadin-binding site(s) in RYR1 affects SR Ca2+ release, alanine-substituted single (D4878A, D4907A, and E4908A) and triple (RYR1-TM) mutants of D4878, D4907, and E4908 were expressed in RYR1-null myotubes. Co-immunoprecipitation experiments showed a 50–60% decrease of triadin brought down in the D4907A and RYR1-TM complexes compared to the triadin–wtRYR1 complex. Ca2+ imaging experiments using Fluo-4-AM showed atypical caffeine responses in myotubes expressing D4907A and RYR1-TM characterized by either a lack of or slower activation and faster inactivation of Ca2+ transients. The results suggest that disruption of interaction between triadin and RYR1 impairs RYR1 function and SR Ca2+ release.
-
amino acid residues gln4020 and lys4021 of the ryanodine receptor type 1 are required for activation by 4 chloro m cresol
Journal of Biological Chemistry, 2006Co-Authors: James D Fessenden, Isaac N. Pessah, Wei Feng, Paul D. AllenAbstract:The ryanodine receptor type 1 (RYR1) and type 2 (RyR2), but not type 3 (RyR3), are efficiently activated by 4-chloro-m-cresol (4-CmC). We previously showed that a 173-amino acid segment of RYR1 (residues 4007-4180) is required for channel activation by 4-CmC (Fessenden, J. D., Perez, C. F., Goth, S., Pessah, I. N., and Allen, P. D. (2003) J. Biol. Chem. 278, 28727-28735). In the present study, we used site-directed mutagenesis to identify individual amino acid(s) within this region that mediate 4-CmC activation. In RYR1, substitution of 11 amino acids conserved between RYR1 and RyR2, but divergent in RyR3, with their RyR3 counterparts reduced 4-CmC sensitivity to the same degree as substitution of the entire 173-amino acid segment. Further analysis of various RYR1 mutants containing successively smaller numbers of these mutations identified 2 amino acid residues (Gln(4020) and Lys(4021)) that, when mutated to their RyR3 counterparts (Leu(3873) and Gln(3874)), abolished 4-CmC activation of RYR1. Mutation of either of these residues alone did not abolish 4-CmC sensitivity, although Q4020L partially reduced 4-CmC-induced Ca(2+) transients. In addition, mutation of the corresponding residues in RyR3 to their RYR1 counterparts (L3873Q/Q3874K) imparted 4-CmC sensitivity to RyR3. Recordings of single RYR1 channels indicated that 4-CmC applied to either the luminal or cytoplasmic side activated the channel with equal potency. Secondary structure modeling in the vicinity of the Gln(4020)-Lys(4021) dipeptide suggests that the region contains a surface-exposed region adjacent to a hydrophobic segment, indicating that both hydrophilic and hydrophobic regions of RYR1 are necessary for 4-CmC binding to the channel and/or to translate allosteric 4-CmC binding into channel activation.
-
Structural characterization of the RYR1-FKBP12 interaction.
Journal of molecular biology, 2005Co-Authors: Montserrat Samso, Xiaohua Shen, Paul D. AllenAbstract:The 12 kDa FK506-binding protein (FKBP12) constitutively binds to the calcium release channel RYR1. Removal of FKBP12 using FK506 or rapamycin causes an increased open probability and an increase in the frequency of sub-conductance states in RYR1. Using cryo-electron microscopy and single-particle image processing, we have determined the 3D difference map of FKBP12 associated with RYR1 at 16 A resolution that can be fitted with the atomic model of FKBP12 in a unique orientation. This has allowed us to better define the surfaces of close apposition between FKBP12 and RYR1. Our results shed light on the role of several FKBP12 residues that had been found critical for the specificity of the RYR1-FKBP12 interaction. As predicted from previous immunoprecipitation studies, our results suggest that Gln3 participates directly in this interaction. The orientation of RYR1-bound FKBP12, with part of its FK506 binding site facing towards RYR1, allows us to propose how FK506 is involved in the dissociation of FKBP12 from RYR1.
Kurt G. Beam - One of the best experts on this subject based on the ideXlab platform.
-
Junctional trafficking and restoration of retrograde signaling by the cytoplasmic RYR1 domain.
The Journal of general physiology, 2017Co-Authors: Alexander Polster, Stefano Perni, Dilyana Filipova, Ong Moua, Joshua D. Ohrtman, Hicham Bichraoui, Kurt G. Beam, Symeon PapadopoulosAbstract:The type 1 ryanodine receptor (RYR1) in skeletal muscle is a homotetrameric protein that releases Ca2+ from the sarcoplasmic reticulum (SR) in response to an "orthograde" signal from the dihydropyridine receptor (DHPR) in the plasma membrane (PM). Additionally, a "retrograde" signal from RYR1 increases the amplitude of the Ca2+ current produced by CaV1.1, the principle subunit of the DHPR. This bidirectional signaling is thought to depend on physical links, of unknown identity, between the DHPR and RYR1. Here, we investigate whether the isolated cytoplasmic domain of RYR1 can interact structurally or functionally with CaV1.1 by producing an N-terminal construct (RYR11:4300) that lacks the C-terminal membrane domain. In CaV1.1-null (dysgenic) myotubes, RYR11:4300 is diffusely distributed, but in RYR1-null (dyspedic) myotubes it localizes in puncta at SR-PM junctions containing endogenous CaV1.1. Fluorescence recovery after photobleaching indicates that diffuse RYR11:4300 is mobile, whereas resistance to being washed out with a large-bore micropipette indicates that the punctate RYR11:4300 stably associates with PM-SR junctions. Strikingly, expression of RYR11:4300 in dyspedic myotubes causes an increased amplitude, and slowed activation, of Ca2+ current through CaV1.1, which is almost identical to the effects of full-length RYR1. Fast protein liquid chromatography indicates that ∼25% of RYR11:4300 in diluted cytosolic lysate of transfected tsA201 cells is present in complexes larger in size than the monomer, and intermolecular fluorescence resonance energy transfer implies that RYR11:4300 is significantly oligomerized within intact tsA201 cells and dyspedic myotubes. A large fraction of these oligomers may be homotetramers because freeze-fracture electron micrographs reveal that the frequency of particles arranged like DHPR tetrads is substantially increased by transfecting RyR-null myotubes with RYR11:4300 In summary, the RYR1 cytoplasmic domain, separated from its SR membrane anchor, retains a tendency toward oligomerization/tetramerization, binds to SR-PM junctions in myotubes only if CaV1.1 is also present and is fully functional in retrograde signaling to CaV1.1.
-
Distinct Components of Retrograde CaV1.1-RYR1 Coupling Revealed by a Lethal Mutation in RYR1
Biophysical journal, 2016Co-Authors: Roger A. Bannister, David C. Sheridan, Kurt G. BeamAbstract:The molecular basis for excitation-contraction coupling in skeletal muscle is generally thought to involve conformational coupling between the L-type voltage-gated Ca(2+) channel (CaV1.1) and the type 1 ryanodine receptor (RYR1). This coupling is bidirectional; in addition to the orthograde signal from CaV1.1 to RYR1 that triggers Ca(2+) release from the sarcoplasmic reticulum, retrograde signaling from RYR1 to CaV1.1 results in increased amplitude and slowed activation kinetics of macroscopic L-type Ca(2+) current. Orthograde coupling was previously shown to be ablated by a glycine for glutamate substitution at RYR1 position 4242. In this study, we investigated whether the RYR1-E4242G mutation affects retrograde coupling. L-type current in myotubes homozygous for RYR1-E4242G was substantially reduced in amplitude (∼80%) relative to that observed in myotubes from normal control (wild-type and/or heterozygous) myotubes. Analysis of intramembrane gating charge movements and ionic tail current amplitudes indicated that the reduction in current amplitude during step depolarizations was a consequence of both decreased CaV1.1 membrane expression (∼50%) and reduced channel Po (∼55%). In contrast, activation kinetics of the L-type current in RYR1-E4242G myotubes resembled those of normal myotubes, unlike dyspedic (RYR1 null) myotubes in which the L-type currents have markedly accelerated activation kinetics. Exogenous expression of wild-type RYR1 partially restored L-type current density. From these observations, we conclude that mutating residue E4242 affects RYR1 structures critical for retrograde communication with CaV1.1. Moreover, we propose that retrograde coupling has two distinct and separable components that are dependent on different structural elements of RYR1.
-
Testing for Direct Interactions Between the DHPR and the RYR1 Cytoplasmic Foot
Biophysical Journal, 2014Co-Authors: Hicham Bichraoui, Alexander Polster, Ong Moua, Tsutomu Tanabe, Simon Papadopoulos, Kurt G. BeamAbstract:In skeletal muscle, RYR1 (5,037 residues) releases calcium from the sarcoplasmic reticulum (SR) in response to an orthograde signal from the DHPR in the plasma membrane (PM), and transmits a retrograde signal which increases the L-type calcium current via the DHPR (which contains CaV1.1 as its principal subunit). Previously, we tested the behavior of a RYR1 construct (YFP-RYR11:4300), which encodes the cytoplasmic domain of RYR1 ("foot") but lacks the C-terminal domains which form the ion pore for SR calcium release and which anchor RYR1 in the SR membrane. We found that YFP-RYR11:4300 targets to PM-SR junctions in dyspedic myotubes (null for endogenous RYR1) and transmits the retrograde signal to the DHPRs present in those myotubes. We have now begun to examine whether these interactions between the RYR1 foot and DHPR can occur in the absence of other proteins which are present in PM-SR junctions of muscle cells. Toward this end, we used tsA201 cells to co-express CFP-RYR11:4300, YFP-CaV1.1 (or CaV1.1/CaV1.2 chimeras) and the DHPR auxiliary subunit β1a. Most of the CaV constructs appeared to be retained in a peri-nuculear compartment (CaV1.1, and chimeras in which three CaV1.1 repeats were replaced by the corresponding CaV1.2 repeats). PM-targeting was observed for a construct composed of CaV1.2 with the cytoplasmic II-III loop replaced by that of CaV1.1. However, we have not yet observed co-localization of CFP-RYR11:4300 with any of the CaV constructs. We are currently testing whether the presence of additional, junctional proteins will result in co-localization of RYR11:4300 and CaV. Supported by grants NIH AR055104 and MDA 277475 to K.G.B.
-
ryanodine receptor type 1 RYR1 mutations c4958s and c4961s reveal excitation coupled calcium entry ecce is independent of sarcoplasmic reticulum store depletion
Journal of Biological Chemistry, 2005Co-Authors: Alanna M Hurne, Kurt G. Beam, Paul D. Allen, Jennifer J Obrien, D E Wingrove, Gennady Cherednichenko, Isaac N. PessahAbstract:Bi-directional signaling between ryanodine receptor type 1 (RYR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RYR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RYR1 conformation. In this report, we substitute RYR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RYR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RYR1 triggers calcium entry via ECCE that resembles that for wild-type RYR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RYR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RYR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RYR1 that influence both its function as a release channel and its interaction with ECCE channels.
-
identification of a region of RYR1 that participates in allosteric coupling with the α1s cav1 1 ii iii loop
Journal of Biological Chemistry, 2002Co-Authors: Catherine Proenza, Paul D. Allen, Jennifer J Obrien, Junichi Nakai, Santwana Mukherjee, Kurt G. BeamAbstract:Abstract In skeletal muscle, excitation-contraction (EC) coupling and retrograde signaling are thought to result from direct interactions between the ryanodine receptor (RYR1) and the α1 subunit of the dihydropyridine receptor (α1S). Previous work has shown that the s53 region of α1S (residues 720–765 in the II–III loop) and regions R10 (1635–2636) and R9 (2659–3720) of RYR1 are involved in this signaling. Using the yeast two-hybrid system, we here report an interaction between s53 and the sR16 region of RYR1 (1837–2168, within R10), whereas no interaction was seen using upstream residues of the α1S II–III loop (s31, 666–709). The specificity of the s53-sR16 interaction was tested by using fragments of the cardiac RyR (RyR2) and DHPR (α1C) that correspond to sR16 and s53, respectively. No interaction was observed for sR16 × c53 (α1C 850–897), but weak interaction was occasionally observed for s53 × cR16 (RyR2 1817–2142). To test the functional significance of the s53 × sR16 interaction, we expressed in dyspedic myotubes a chimeric RyR (chimeraR16) in which sR16 was substituted for the corresponding region of RyR2. ChimeraR16 was found to mediate weak skeletal-type EC coupling. To test the necessity of sR16 sequence for coupling, we used “chimeraR16-rev,” in which sR16 and a small upstream region of RYR1 were replaced by RyR2 sequence. ChimeraR16-rev did not differ from RYR1 in its ability to mediate EC coupling. Thus, interaction between residues 720–765 of α1S and residues 1837–2168 of RYR1 appears to contribute to but is not essential for EC coupling in skeletal muscle.
Isaac N. Pessah - One of the best experts on this subject based on the ideXlab platform.
-
RYR1 mediated ca2 leak and ca2 entry determine resting intracellular ca2 in skeletal myotubes
Journal of Biological Chemistry, 2010Co-Authors: Jose M Eltit, Paul D. Allen, Isaac N. Pessah, Tianzhong Yang, Tadeusz F Molinski, Jose R LopezAbstract:The control of resting free Ca(2+) in skeletal muscle is thought to be a balance of channels, pumps, and exchangers in both the sarcolemma and sarcoplasmic reticulum. We explored these mechanisms using pharmacologic and molecular perturbations of genetically engineered (dyspedic) muscle cells that constitutively lack expression of the skeletal muscle sarcoplasmic reticulum Ca(2+) release channels, RYR1 and RyR3. We demonstrate here that expression of RYR1 is responsible for more than half of total resting Ca(2+) concentration ([Ca(2+)](rest)) measured in wild type cells. The elevated [Ca(2+)](rest) in RYR1-expressing cells is not a result of active gating of the RYR1 channel but instead is accounted for by the RYR1 ryanodine-insensitive Ca(2+) leak conformation. In addition, we demonstrate that basal sarcolemmal Ca(2+) influx is also governed by RYR1 expression and contributes in the regulation of [Ca(2+)](rest) in skeletal myotubes.
-
enantiomeric specificity of 2 2 3 3 6 6 hexachlorobiphenyl toward ryanodine receptor types 1 and 2
Chemical Research in Toxicology, 2009Co-Authors: Isaac N. Pessah, Claudio F. Perez, Hansjoachim Lehmler, Larry W Robertson, Elaine Cabrales, Diptiman D Bose, Wei FengAbstract:Polychlorinated biphenyls (PCBs) with unsymmetrical chlorine substitutions and multiple ortho-substitutions that restrict rotation around the biphenyl bond may exist in two stable enantiomeric forms. Stereospecific binding and functional modification of specific biological signaling targets have not been previously described for PCB atropisomers. We report that (−)-2,2′,3,3′,6,6′-hexachlorobiphenyl [(−)-PCB 136] enhances the binding of [3H]ryanodine to high-affinity sites on ryanodine receptors type 1 (RYR1) and type 2 (RyR2) (EC50 values ∼0.95 μM), whereas (+)-PCB 136 is inactive at ≤10 μM. (−)-PCB 136 induces a rapid release of Ca2+ from microsomal vesicles by selective sensitization of RyRs, an effect not antagonized by (+)-PCB 136. (−)-PCB 136 (500nM) enhances the activity of reconstituted RYR1 channels 3-fold by stabilizing the open and destabilizing the closed conformational states. The enantiomeric specificity is also demonstrated in intact HEK 293 cells expressing RYR1 where exposure to (−)-PCB 13...
-
amino acid residues gln4020 and lys4021 of the ryanodine receptor type 1 are required for activation by 4 chloro m cresol
Journal of Biological Chemistry, 2006Co-Authors: James D Fessenden, Isaac N. Pessah, Wei Feng, Paul D. AllenAbstract:The ryanodine receptor type 1 (RYR1) and type 2 (RyR2), but not type 3 (RyR3), are efficiently activated by 4-chloro-m-cresol (4-CmC). We previously showed that a 173-amino acid segment of RYR1 (residues 4007-4180) is required for channel activation by 4-CmC (Fessenden, J. D., Perez, C. F., Goth, S., Pessah, I. N., and Allen, P. D. (2003) J. Biol. Chem. 278, 28727-28735). In the present study, we used site-directed mutagenesis to identify individual amino acid(s) within this region that mediate 4-CmC activation. In RYR1, substitution of 11 amino acids conserved between RYR1 and RyR2, but divergent in RyR3, with their RyR3 counterparts reduced 4-CmC sensitivity to the same degree as substitution of the entire 173-amino acid segment. Further analysis of various RYR1 mutants containing successively smaller numbers of these mutations identified 2 amino acid residues (Gln(4020) and Lys(4021)) that, when mutated to their RyR3 counterparts (Leu(3873) and Gln(3874)), abolished 4-CmC activation of RYR1. Mutation of either of these residues alone did not abolish 4-CmC sensitivity, although Q4020L partially reduced 4-CmC-induced Ca(2+) transients. In addition, mutation of the corresponding residues in RyR3 to their RYR1 counterparts (L3873Q/Q3874K) imparted 4-CmC sensitivity to RyR3. Recordings of single RYR1 channels indicated that 4-CmC applied to either the luminal or cytoplasmic side activated the channel with equal potency. Secondary structure modeling in the vicinity of the Gln(4020)-Lys(4021) dipeptide suggests that the region contains a surface-exposed region adjacent to a hydrophobic segment, indicating that both hydrophilic and hydrophobic regions of RYR1 are necessary for 4-CmC binding to the channel and/or to translate allosteric 4-CmC binding into channel activation.
-
ryanodine receptor type 1 RYR1 mutations c4958s and c4961s reveal excitation coupled calcium entry ecce is independent of sarcoplasmic reticulum store depletion
Journal of Biological Chemistry, 2005Co-Authors: Alanna M Hurne, Kurt G. Beam, Paul D. Allen, Jennifer J Obrien, D E Wingrove, Gennady Cherednichenko, Isaac N. PessahAbstract:Bi-directional signaling between ryanodine receptor type 1 (RYR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RYR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RYR1 conformation. In this report, we substitute RYR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RYR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RYR1 triggers calcium entry via ECCE that resembles that for wild-type RYR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RYR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RYR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RYR1 that influence both its function as a release channel and its interaction with ECCE channels.
-
RYR1/RyR3 chimeras reveal that multiple domains of RYR1 are involved in skeletal-type E-C coupling.
Biophysical journal, 2003Co-Authors: Claudio F. Perez, Andrew A. Voss, Isaac N. Pessah, Paul D. AllenAbstract:Skeletal-type E-C coupling is thought to require a direct interaction between RYR1 and the α1S-DHPR. Most available evidence suggests that the cytoplasmic II–III loop of the dihydropyridine receptor (DHPR) is the primary source of the orthograde signal. However, identification of the region(s) of RYR1 involved in bidirectional signaling with the α1S-DHPR remains elusive. To identify these regions we have designed a series of chimeric RyR cDNAs in which different segments of RYR1 were inserted into the corresponding region of RyR3 and expressed in dyspedic 1B5 myotubes. RyR3 provides a preferable background than RyR2 for defining domains essential for E-C coupling because it possesses less sequence homology to RYR1 than the RyR2 backbone used in previous studies. Our data show that two regions of RYR1 (chimera Ch-10 aa 1681–2641 and Ch-9 aa 2642–3770), were independently able to restore skeletal-type E-C coupling to RyR3. These two regions were further mapped and the critical RYR1 residues were 1924–2446 (Ch-21) and 2644–3223 (Ch-19). These results both support and refine the previous hypothesis that multiple domains of RYR1 combine to functionally interact with the DHPR during E-C coupling.
Robert T. Dirksen - One of the best experts on this subject based on the ideXlab platform.
-
reduced threshold for store overload induced ca2 release is a common defect of RYR1 mutations associated with malignant hyperthermia and central core disease
Biochemical Journal, 2017Co-Authors: Wenqian Chen, Ruiwu Wang, Andrea Koop, Robert T. Dirksen, David H. Maclennan, Yingjie Liu, Wenting Guo, Jinhong Wei, Sui Rong Wayne ChenAbstract:Mutations in the skeletal muscle ryanodine receptor (RYR1) cause malignant hyperthermia (MH) and central core disease (CCD), whereas mutations in the cardiac ryanodine receptor (RyR2) lead to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most disease-associated RYR1 and RyR2 mutations are located in the N-terminal, central, and C-terminal regions of the corresponding ryanodine receptor (RyR) isoform. An increasing body of evidence demonstrates that CPVT-associated RyR2 mutations enhance the propensity for spontaneous Ca2+ release during store Ca2+ overload, a process known as store overload-induced Ca2+ release (SOICR). Considering the similar locations of disease-associated RYR1 and RyR2 mutations in the RyR structure, we hypothesize that like CPVT-associated RyR2 mutations, MH/CCD-associated RYR1 mutations also enhance SOICR. To test this hypothesis, we determined the impact on SOICR of 12 MH/CCD-associated RYR1 mutations E2347-del, R2163H, G2434R, R2435L, R2435H, and R2454H located in the central region, and Y4796C, T4826I, L4838V, A4940T, G4943V, and P4973L located in the C-terminal region of the channel. We found that all these RYR1 mutations reduced the threshold for SOICR. Dantrolene, an acute treatment for MH, suppressed SOICR in HEK293 cells expressing the RYR1 mutants R164C, Y523S, R2136H, R2435H, and Y4796C. Interestingly, carvedilol, a commonly used β-blocker that suppresses RyR2-mediated SOICR, also inhibits SOICR in these RYR1 mutant HEK293 cells. Therefore, these results indicate that a reduced SOICR threshold is a common defect of MH/CCD-associated RYR1 mutations, and that carvedilol, like dantrolene, can suppress RYR1-mediated SOICR. Clinical studies of the effectiveness of carvedilol as a long-term treatment for MH/CCD or other RYR1-associated disorders may be warranted.
-
Novel Excitation-Contraction Uncoupled RYR1 Mutations in Patients With Central Core Disease
Neuromuscular disorders : NMD, 2012Co-Authors: Natalia Kraeva, Elena Zvaritch, Ann E. Rossi, Sanjeewa A. Goonasekera, Hilal Zaid, Wanda Frodis, Alexander Kraev, Robert T. Dirksen, David H. Maclennan, Sheila RiaziAbstract:Abstract Central core disease, one of the most common congenital myopathies in humans, has been linked to mutations in the RYR1 gene encoding the Ca 2+ release channel of the sarcoplasmic reticulum (RYR1). Functional analyses showed that disease-associated RYR1 mutations led to impairment of skeletal muscle Ca 2+ homeostasis; however, thorough understanding of the molecular mechanisms underlying central core disease and other RYR1-related conditions is still lacking. We screened by sequencing the complete RYR1 transcripts in ten unrelated patients with central core disease and identified five novel, p.M4640R, p.L4647P, p.F4808L, p.D4918N and p.F4941C, and four recurrent mutations. Four of the novel mutations involved amino acid residues that were positioned within putative transmembrane segments of the RYR1. The pathogenic character of the identified mutations was demonstrated by bioinformatic analyses and by the in vitro functional studies in HEK293 cells and RYR1-null (dyspedic) myotubes. Characterization of Ca 2+ channel properties of RYR1s carrying one recurrent and two novel mutations upholds the view that diminished intracellular Ca 2+ release caused by impaired Ca 2+ channel gating and/or Ca 2+ permeability is an important component of central core disease etiology. This study expands the list of functionally characterized disease-associated RYR1 mutations, increasing the value of genetic diagnosis for RYR1-related disorders.
-
Defects in Ca2+ release associated with local expression of pathological ryanodine receptors in mouse muscle fibres.
The Journal of Physiology, 2011Co-Authors: Romain Lefebvre, Robert T. Dirksen, Claude Legrand, Estela González-rodríguez, Linda Groom, Vincent JacquemondAbstract:Mutations of the gene encoding the type 1 ryanodine receptor (RYR1) are associated with skeletal muscle disorders including malignant hyperthermia susceptibility (MHS) and central core disease (CCD). We used in vivo expression of EGFP-RYR1 constructs in fully differentiated mouse muscle fibres to characterize the function of several RYR1 mutants. Wild-type and Y523S, R615C, R2163H and I4897T mutants of RYR1 were separately expressed and found to be present within restricted regions of fibres with a pattern consistent with triadic localization. Confocal measurements of voltage-clamp-activated myoplasmic Ca(2+) transients demonstrated alterations of sarcoplasmic reticulum (SR) Ca(2+) release spatially correlated with the presence of exogenous RYR1s. The Y523S, R615C and R2163H RYR1 MHS-related mutants were associated with enhanced peak Ca(2+) release for low and moderate levels of depolarization, whereas the I4897T CCD mutant produced a chronic reduction of peak SR Ca(2+) release. For example, peak Ca(2+) release in response to a depolarization to -20 mV in regions of fibres expressing Y523S and I4897T was 2.0 ± 0.3 (n = 9) and 0.46 ± 0.1 (n = 5) times the corresponding value in adjacent, non-expressing regions of the same fibre, respectively. Interestingly no significant change in the estimated total amount of Ca(2+) released at the end of large depolarizing pulses was observed for any of the mutant RYR1 channels. Overall, results are consistent with an 'inherent' increase in RYR1 sensitivity to activation by the voltage sensor for the MHS-related RYR1 mutants and a partial failure of voltage-gated release for the CCD-related I4897T mutant, that occur with no sign of change in SR Ca(2+) content. Furthermore, the results indicate that RYR1 channel density is tightly regulated even under the present conditions of forced exogenous expression.
-
The elusive role of the SPRY2 domain in RYR1
Channels (Austin Tex.), 2011Co-Authors: Hanshen Tae, Marco G Casarotto, Philip G. Board, Robert T. Dirksen, Lan Wei, Esther M. Gallant, Hermia Willemse, Shamaruh Mirza, Angela F. DulhuntyAbstract:The second of three SPRY domains (SPRY2, S1085 -V1208) located in the skeletal muscle ryanodine receptor (RYR1) is contained within regions of RYR1 that influence EC coupling and bind to imperatoxin A, a toxin probe of RYR1 channel gating. We examined the binding of the F loop (P1107-A1121) in SPRY2 to the ASI/basic region in RYR1 (T3471-G3500, containing both alternatively spliced (ASI) residues and neighboring basic amino acids). We then investigated the possible influence of this interaction on excitation contraction (EC) coupling. A peptide with the F loop sequence and an antibody to the SPRY2 domain each enhanced RYR1 activity at low concentrations and inhibited at higher concentrations. A peptide containing the ASI/basic sequence bound to SPRY2 and binding decreased ~10-fold following mutation or structural disruption of the basic residues. Binding was abolished by mutation of three critical acidic F loop residues. Together these results suggest that the ASI/basic and SPRY2 domains interact in an F loop regulatory module. Although a region that includes the SPRY2 domain influences EC coupling, as does the ASI/basic region, Ca2+ release during ligand- and depolarization-induced RYR1 activation were not altered by mutation of the three critical F loop residues following expression of mutant RYR1 in RYR1-null myotubes. Therefore the electrostatic regulatory interaction between the SPRY2 F loop residues (that bind to imperatoxin A) and the ASI/basic residues of RYR1 does not influence bi-directional DHPR-RYR1 signaling during skeletal EC coupling, possibly because the interaction is interrupted by the influence of factors present in intact muscle cells.
-
Alternative splicing of RYR1 alters the efficacy of skeletal EC coupling.
Cell calcium, 2009Co-Authors: Takashi Kimura, Marco G Casarotto, Noriaki Ikemoto, Robert T. Dirksen, John D. Lueck, Peta J. Harvey, Suzy M. Pace, Angela F. DulhuntyAbstract:Alternative splicing of ASI residues (Ala(3481)-GIn(3485)) in the skeletal muscle ryanodine receptor (RYR1) is developmentally regulated: the residues are present in adult ASI(+)RYR1, but absent in the juvenile ASI(-)RYR1 which is over-expressed in adult myotonic dystrophy type 1 (DM1). Although this splicing switch may influence RYR1 function in developing muscle and DM1, little is known about the properties of the splice variants. We examined excitation-contraction (EC) coupling and the structure and interactions of the ASI domain (Thr(3471)-Gly(3500)) in the splice variants. Depolarisation-dependent Ca(2+) release was enhanced by >50% in myotubes expressing ASI(-)RYR1 compared with ASI(+)RYR1, although DHPR L-type currents and SIR Ca(2+) content were unaltered, while ASI(-)RYR1 channel function was actually depressed. The effect on EC coupling did not depend on changes in ASI domain secondary structure. Probing RYR1 function with peptides possessing the ASI domain sequence indicated that the domain contributes to an inhibitory module in RYR1. The action of the peptide depended on a sequence of basic residues and their alignment in an alpha-helix adjacent to the ASI splice site. This is the first evidence that the ASI residues contribute to an inhibitory module in RYR1 that influences EC coupling. Implications for development and DM1 are discussed. (c) 2008 Elsevier Ltd. All rights reserved.