ORCID Profile
0000-0002-7616-500X
Current Organisations
University of New South Wales
,
University of Turku
,
University of Bergen
,
Australian National University
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Publisher: Springer Science and Business Media LLC
Date: 15-08-2006
DOI: 10.1007/S10974-006-9086-1
Abstract: There are many mutations in the ryanodine receptor (RyR) Ca2+ release channel that are implicated in skeletal muscle disorders and cardiac arrhythmias. More than 80 mutations in the skeletal RyR1 have been identified and linked to malignant hyperthermia, central core disease or multi-minicore disease, while more than 40 mutations in the cardiac RyR2 lead to ventricular arrhythmias and sudden cardiac death in patients with structurally normal hearts. These RyR mutations cause erse changes in RyR activity which either excessively activate or block the channel in a manner that disrupts Ca2+ signalling in the muscle fibres. In a different myopathy, myotonic dystrophy (DM), a juvenile isoform of the skeletal RyR is preferentially expressed in adults. There are two regions of RyR1 that are variably spiced and developmentally regulated (ASI and ASII). The juvenile isoform (ASI(-)) is less active than the adult isoform (ASI(+)) and its over-expression in adults with DM may contribute to functional changes. Finally, mutations in an important regulator of the RyR, the Ca2+ binding protein calsequestrin (CSQ), have been linked to a disruption of Ca2+ homeostasis in cardiac myocytes that results in arrhythmias. We discuss evidence supporting the hypothesis that mutations in each of these situations alter protein rotein interactions within the RyR complex or between the RyR and its associated proteins. The disruption of these protein-protein interactions can lead either to excess Ca2+ release or reduced Ca2+ release and thus to abnormal Ca2+ homeostasis. Much of the evidence for disruption of protein-protein interactions has been provided by the actions of a group of novel RyR regulators, domain peptides with sequences that correspond to sequences within the RyR and which compete with the endogenous residues for their interaction sites.
Publisher: American Physiological Society
Date: 03-2018
DOI: 10.1152/AJPCELL.00187.2017
Abstract: Malignant hyperthermia (MH) susceptibility has been recently linked to a novel variant of β 1a subunit of the dihydropyridine receptor (DHPR), a channel essential for Ca 2+ regulation in skeletal muscle. Here we evaluate the effect of the mutant variant V156A on the structure/function of DHPR β 1a subunit and assess its role on Ca 2+ metabolism of cultured myotubes. Using differential scanning fluorimetry, we show that mutation V156A causes a significant reduction in thermal stability of the Src homology 3/guanylate kinase core domain of β 1a subunit. Expression of the variant subunit in β 1 -null mouse myotubes resulted in increased sensitivity to caffeine stimulation. Whole cell patch-cl analysis of β 1a -V156A-expressing myotubes revealed a −2 mV shift in voltage dependence of channel activation, but no changes in Ca 2+ conductance, current kinetics, or sarcoplasmic reticulum Ca 2+ load were observed. Measurement of resting free Ca 2+ and Na + concentrations shows that both cations were significantly elevated in β 1a -V156A-expressing myotubes and that these changes were linked to increased rates of plasmalemmal Ca 2+ entry through Na + /Ca 2+ exchanger and/or transient receptor potential canonical channels. Overall, our data show that mutant variant V156A results in instability of protein subdomains of β 1a subunit leading to a phenotype of Ca 2+ dysregulation that partly resembles that of other MH-linked mutations of DHPR α 1S subunit. These data prove that homozygous expression of variant β 1a -V156A has the potential to be a pathological variant, although it may require other gene defects to cause a full MH phenotype.
Publisher: Wiley
Date: 03-1978
DOI: 10.1113/JPHYSIOL.1978.SP012220
Abstract: 1. The steady-state intracellular membrane potential of fibres in thin bundles dissected from mouse extensor digitorum longus or soleus muscles or rat sternomastoid muscles was measured with 3 M-KCl glass micro-electrodes. The steady-state membrane potential was found to depend on the extracellular concentrations of Na, K and Cl ions. 2. The resting membrane potential (3.5 mM-[K]o, 160 mM-[Cl]o) was -74 +/- 1 mV (mean +/- S.E.) and a reduction in [Cl]o to 3.5 mM caused a reversible steady-state hyperpolarization to -94 +/- 1 mV (mean +/- S.E.). 3. The steady-state membrane potentials recorded in fibres exposed to different [K]o and zero [Cl]o were consistent with potentials predicted by the Goldman, Hodgkin & Katz (GHK) equation for Na and K. The results of similar experiments done with Cl as the major external anion could not be fitted by the same equation. 4. The GHK equation for Na, K and Cl did fit data obtained from fibres in solutions containing different [K]o with Cl as the major external anion if the intracellular Cl concentration was allowed to be out of equilibrium with the steady-state membrane potential. 5. It is suggested that an active influx of Cl ions controls the intracellular Cl concentrations in these fibres and hence maintains the Cl equilibrium potential at a depolarized value with respect to the resting membrane potential. 6. The steady-state membrane potential of rat diaphragm fibres was independent of [Cl]o and it seems likely that the intracellular Cl concentration of these fibres is not controlled by active Cl transport.
Publisher: Elsevier BV
Date: 10-2008
DOI: 10.1016/J.CECA.2008.01.005
Abstract: Calcium signaling, intrinsic to skeletal and cardiac muscle function, is critically dependent on the amount of calcium stored within the sarcoplasmic reticulum. Calsequestrin, the main calcium buffer in the sarcoplasmic reticulum, provides a pool of calcium for release through the ryanodine receptor and acts as a luminal calcium sensor for the channel via its interactions with triadin and junctin. We examined the influence of phosphorylation of calsequestrin on its ability to store calcium, to polymerise and to regulate ryanodine receptors by binding to triadin and junctin. Our hypothesis was that these parameters might be altered by phosphorylation of threonine 353, which is located near the calcium and triadin/junctin binding sites. Although phosphorylation increased the calcium binding capacity of calsequestrin nearly 2-fold, it did not alter calsequestrin polymerisation, its binding to triadin or junctin or inhibition of ryanodine receptor activity at 1 mM luminal calcium. Phosphorylation was required for calsequestrin binding to junctin when calcium concentration was low (100 nM), and ryanodine receptors were activated by dephosphorylated calsequestrin when it bound to triadin alone. These novel data shows that phosphorylated calsequestrin is required for high capacity calcium buffering and suggest that ryanodine receptor inhibition by calsequestrin is mediated by junctin.
Publisher: Springer Science and Business Media LLC
Date: 12-1992
DOI: 10.1007/BF01738254
Publisher: Elsevier BV
Date: 10-2015
DOI: 10.1016/J.BCP.2015.08.004
Abstract: Release of Ca(2+) from the sarcoplasmic reticulum (SR) through the cardiac ryanodine receptor (RyR2) is an essential step in cardiac excitation-contraction coupling. Excess Ca(2+) release due to overactive RyR2 can cause arrhythmia that can lead to cardiac arrest. Fragments derived from the carboxy-terminal domain of human glutathione transferase M2 (GSTM2C) specifically inhibit RyR2 activity. Our aim was to further improve this inhibition by mutagenesis and to assess the therapeutic potential of GSTM2C based peptides to treat Ca(2+) release-based arrhythmia. We generated several mutant variants of the C-terminal fragment GSTM2C H5-8 and from those mutant proteins we identified two (RM13 and SM2) that exhibited significantly greater inhibition of cardiac SR Ca(2+) release and single RyR2 channel activity. Flow cytometry analysis showed that these two mutant proteins as well as GSTM2C H5-8 are taken up by isolated adult mouse cardiomyocytes without the aid of any additional compounds, Ca(2+) imaging and isolated cell contraction measurements revealed that GSTM2C H5-8, SM2 and RM13 reduce the SR Ca(2+) release rate and the fractional shortening of adult mouse cardiomyocytes, while importantly increasing the rate of Ca(2+) removal from the sarcoplasm. These observations indicate that peptides derived from GSTM2C inhibit RyR2 at a cellular level and thus they may provide the basis for a novel therapeutic agent to treat arrhythmia and heart attack.
Publisher: Springer Science and Business Media LLC
Date: 12-1995
DOI: 10.1007/BF00130241
Abstract: Aflatoxin M1 (AFM1) is the only mycotoxin with maximum residue limit in milk, which may result in serious human diseases. On the contrary, lactoferrin (Lf) is an active protein with multiple functions. Studies have confirmed that Lf has a powerful potential to protect the intestines, but the influence of Lf on mycotoxins is not clear. This study aims to explore whether Lf can protect the cytotoxicity induced by AFM1, and determine the underlying mechanisms in human normal colonic epithelial NCM460 cells. The results indicated that AFM1 decreased the cell viability, and increased the levels of apoptosis and autophagy of NCM460 cells. Lf can alleviate the cytotoxicity induced by AFM1 through enhancing cell viability, significantly down-regulated the expression of apoptotic genes and proteins (
Publisher: Proceedings of the National Academy of Sciences
Date: 13-05-2013
Abstract: We recently reported the isolation of a scorpion toxin named U 1 -liotoxin-Lw1a (U 1 -LITX-Lw1a) that adopts an unusual 3D fold termed the disulfide-directed hairpin (DDH) motif, which is the proposed evolutionary structural precursor of the three-disulfide-containing inhibitor cystine knot (ICK) motif found widely in animals and plants. Here we reveal that U 1 -LITX-Lw1a targets and activates the mammalian ryanodine receptor intracellular calcium release channel (RyR) with high (fM) potency and provides a functional link between DDH and ICK scorpion toxins. Moreover, U 1 -LITX-Lw1a, now described as φ-liotoxin-Lw1a (φ-LITX-Lw1a), has a similar mode of action on RyRs as scorpion calcines, although with significantly greater potency, inducing full channel openings at lower (fM) toxin concentrations whereas at higher pM concentrations increasing the frequency and duration of channel openings to a submaximal state. In addition, we show that the C-terminal residue of φ-LITX-Lw1a is crucial for the increase in full receptor openings but not for the increase in receptor subconductance opening, thereby supporting the two-binding-site hypothesis of scorpion toxins on RyRs. φ-LITX-Lw1a has potential both as a pharmacological tool and as a lead molecule for the treatment of human diseases that involve RyRs, such as malignant hyperthermia and polymorphic ventricular tachycardia.
Publisher: Elsevier BV
Date: 05-2002
DOI: 10.1016/S0079-6107(02)00013-5
Abstract: Excitation-contraction coupling in both skeletal and cardiac muscle depends on structural and functional interactions between the voltage-sensing dihydropyridine receptor L-type Ca(2+) channels in the surface/transverse tubular membrane and ryanodine receptor Ca(2+) release channels in the sarcoplasmic reticulum membrane. The channels are targeted to either side of a narrow junctional gap that separates the external and internal membrane systems and are arranged so that bi-directional structural and functional coupling can occur between the proteins. There is strong evidence for a physical interaction between the two types of channel protein in skeletal muscle. This evidence is derived from studies of excitation-contraction coupling in intact myocytes and from experiments in isolated systems where fragments of the dihydropyridine receptor can bind to the ryanodine receptors in sarcoplasmic reticulum vesicles or in lipid bilayers and alter channel activity. Although micro-regions that participate in the functional interactions have been identified in each protein, the role of these regions and the molecular nature of the protein-protein interaction remain unknown. The trigger for Ca(2+) release through ryanodine receptors in cardiac muscle is a Ca(2+) influx through the L-type Ca(2+) channel. The Ca(2+) entering through the surface membrane Ca(2+) channels flows directly onto underlying ryanodine receptors and activates the channels. This was thought to be a relatively simple system compared with that in skeletal muscle. However, complexities are emerging and evidence has now been obtained for a bi-directional physical coupling between the proteins in cardiac as well as skeletal muscle. The molecular nature of this coupling remains to be elucidated.
Publisher: Elsevier BV
Date: 07-1989
DOI: 10.1016/S0006-3495(89)82647-5
Abstract: New World monkeys of the genus Aotus synthesize a fusion protein (AoT5Cyp) containing tripartite motif-containing 5 (TRIM5) and cyclophilin A (CypA) that potently blocks HIV-1 infection. We attempted to generate a human HIV-1 inhibitor modeled after AoT5Cyp, by fusing human CypA to human TRIM5 (hT5Cyp). Of 13 constructs, 3 showed substantial HIV-1-inhibitory activity when expressed in human cell lines. This activity required capsid binding by CypA and correlated with CypA linkage to the TRIM5a capsid-specificity determinant and the ability to form cytoplasmic bodies. CXCR4- and CCR5-tropic HIV-1 clones and primary isolates were inhibited from infecting multiple human macrophage and T cell lines and primary cells by hT5Cyp, as were HIV-2ROD, SIVAGMtan, FIVPET, and a circulating HIV-1 isolate previously reported to be AoT5Cyp resistant. The anti-HIV-1 activity of hT5Cyp was surprisingly more effective than that of the well-characterized rhesus TRIM5alpha, especially in T cells. hT5Cyp also blocked HIV-1 infection of primary CD4+ T cells and macrophages and conferred a survival advantage to these cells without disrupting their function. Extensive attempts to elicit HIV-1 resistance to hT5Cyp were unsuccessful. Finally, Rag2-/-gammac-/- mice were engrafted with human CD4+ T cells that had been transduced by optimized lentiviral vectors bearing hT5Cyp. Upon challenge with HIV-1, these mice showed decreased viremia and productive infection in lymphoid organs and preserved numbers of human CD4+ T cells. We conclude that hT5Cyp is an extraordinarily robust inhibitor of HIV-1 replication and a promising anti-HIV-1 gene therapy candidate.
Publisher: Springer Science and Business Media LLC
Date: 2000
Abstract: We show that rabbit skeletal RyR channels in lipid bilayers can be activated or inhibited by NO, in a manner that depends on donor concentration, membrane potential and the presence of channel agonists. 10 microm S-nitroso-N-acetyl-penicillamine (SNAP) increased RyR activity at -40 mV within 15 sec of addition to the cis chamber, with a 2-fold increase in frequency of channel opening (F(o)). 10 microm SNAP did not alter activity at +40 mV and did not further activate RyRs previously activated by 2 mm cis ATP at +40 or -40 mV. In contrast to the increase in F(o) with 10 microm SNAP, 1 mm SNAP caused a 2-fold reduction in F(o) but a 1.5-fold increase in mean open time (T(o)) at -40 mV in the absence of ATP. 1 mm SNAP or 0.5 mm sodium nitroprusside (SNP) induced approximately 3-fold reductions in F(o) and T(o) at +40 or -40 mV when channels were activated by 2 mm cis ATP or in channels activated by 6.5 microm peptide A at -40 mV (peptide A corresponds to part of the II-III loop of the skeletal dihydropyridine receptor). Both SNAP-induced activation and SNAP/SNP-induced inhibition were reversed by 2 mm dithiothreitol. The results suggest that S-Nitrosylation or oxidation of at least three classes of protein thiols by NO each produced characteristic changes in RyR activity. We propose that, in vivo, initial release of NO activates RyRs, but stronger release increases [NO] and inhibits RyR activity and contraction.
Publisher: Elsevier BV
Date: 02-2001
Publisher: Elsevier BV
Date: 12-1981
DOI: 10.1016/0304-3940(81)90442-0
Abstract: The aim of this study was to examine the changing influence over time of comorbid heart disease on symptoms and health status in patients with COPD. This is a prospective cohort study of 495 COPD patients with a baseline in 2005 and follow-up in 2012. The study population was ided into three groups: patients without heart disease (no-HD), those diagnosed with heart disease during the study period (new-HD) and those with heart disease at baseline (HD). Symptoms were measured using the mMRC. Health status was measured using the Clinical COPD Questionnaire (CCQ) and the COPD Assessment Test (CAT only available in 2012). Logistic regression with mMRC ≥2 and linear regression with CCQ and CAT scores in 2012 as dependent variables were performed unadjusted, adjusted for potential confounders, and additionally adjusted for baseline mMRC, respectively, CCQ scores. Mean mMRC worsened from 2005 to 2012 as follows: for the no-HD group from 1.8 (±1.3) to 2.0 (±1.4), ( Heart disease contributes to lower health status and higher symptom burden in COPD but does not accelerate the worsening over time.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 22-06-2011
Abstract: Our aim was to examine the molecular basis for acute effects of the anthracycline daunorubicin on cardiac ryanodine receptor (RyR2) channels and cardiac calsequestrin (CSQ2). Cardiotoxic effects of anthracyclines preclude their chemotherapeutic use in patients with pre-existing heart conditions. To address this significant problem, the mechanisms of anthracycline toxicity must be defined but at present are poorly understood. RyR2 channel activity was assessed by measuring Ca²⁺ release from cardiac sarcoplasmic reticulum vesicles and by examining single RyR2 channels inserted into artificial lipid bilayers. We show that 0.5 to 10 μM daunorubicin increases the activity of RyR2 channels after 5 to 10 min and that activity then declines to very low levels when channels are exposed to daunorubicin concentrations of ≥ 2.5 μM for a further 10 to 20 min. Extensive dissection of these effects shows for the first time that the activation results from a redox-independent binding of daunorubicin to the RyR2 complex. Novel data include the demonstration of daunorubicin binding to RyR2. We provide compelling evidence that RyR2 channel inhibition is due to the oxidation of free SH groups. The oxidation reaction is prevented by the presence of 1 mM dithiothreitol. We also present novel data showing that CSQ2 modifies the response of RyR2 to daunorubicin, but that the response of RyR2 is not dependent on daunorubicin binding to CSQ2. We suggest that binding of daunorubicin to RyR2 and CSQ2, and oxidation of RyR2, are all likely to contribute to anthracycline-induced cardiotoxicity during chemotherapy.
Publisher: Springer Science and Business Media LLC
Date: 03-1994
DOI: 10.1007/BF00232792
Abstract: Upper tract urothelial carcinoma (UTUC) has a high recurrence rate and is likely refractory to systemic chemotherapy. The long-term outcomes and responses to immunotherapy and retreatment regimen after tumor recurrence for such cases had not yet been well-documented. Here we report a unique case of long-term follow-up with a 67-year-old woman, who was diagnosed with advanced UTUC, received radical nephroureterectomy with bladder cuff, and was refractory to chemotherapy with cisplatin and gemcitabine. Positive PD-L1 expression and somatic mutation of Ser249Cys in FGFR3 were identified in the tumor tissue. The patient then received pembrolizumab monotherapy and achieved complete response (CR) after 6 cycles of treatment. She discontinued pembrolizumab treatment thereafter but remained in CR for 3 years and 7 months until the recurrence of tumor in the right mid-ureter. The patient was then retreated with a combination of pembrolizumab and erdafitinib, and achieved CR again after the third cycle of treatment. We reported here a rare case of UTUC with concurrent pathogenic mutations in FGFR3 and TP53 with positive PD-L1 expression. The patient archived exceptional therapeutic responses to PD-1 blockade treatment and retreatment with combination of pembrolizumab and erdafitinib. Our results provide new insight into the duration of immunotherapy and the retreatment strategy after tumor recurrence based on in idual genomic profiles.
Publisher: Elsevier BV
Date: 03-2009
Publisher: Rockefeller University Press
Date: 02-1982
DOI: 10.1085/JGP.79.2.233
Abstract: The effect of subthreshold depolarization on mechanical threshold was investigated in tetrodotoxin-poisoned mammalian and hibian skeletal muscle fibers using a two-microelectrode voltage-cl technique. Mechanical threshold was determined with a 2-ms test pulse. The immediate effect of depolarization was inhibition of the mechanical system. The consequent increase in the test pulse threshold was linearly related to the size of the depolarization and there was, on the average, a 10% increase in threshold for a 10-mV depolarization in mammalian fibers. The duration of the inhibitory period was also related to the size of the depolarization. Inhibition was interrupted by the onset of activation (seen as a reduction in the test pulse threshold), and in rat soleus fibers this occurred within 100 ms with a 20-mV depolarization, inhibition decayed within 10 ms. The decay of activation after brief conditioning pulses was initially rapid (on the average, the test pulse threshold recovered to 80% of its control value within 1 ms) and then slow (full recovery took 100-500 ms). After long conditioning pulses, activation often decayed into a period of inhibition. When depolarization (of 20 mV or more) was maintained for several seconds, the fibers became inactivated. Rat extensor digitorum longus and sternomastoid fibers were strongly inactivated by depolarization to -40 mV and the test pulse to +40 mV did not cause contraction.
Publisher: MDPI AG
Date: 29-05-2018
DOI: 10.3390/ARTS7020019
Publisher: Elsevier BV
Date: 07-1983
DOI: 10.1016/S0022-5320(83)90085-0
Abstract: Indentations in the terminal cisternae were measured in freeze-fracture replicas of extensor digitorum longus (EDL) and soleus muscles from normal rats and from rats in which the spinal cord had been transected at the midthoracic level 6 weeks earlier. The density of indentations was compared with contraction time and characteristics of K-contractures. In paraplegic rats, the average soleus contraction time decreased from 89 to 46 msec and the average number of indentations, per micrometer of terminal cisternae, increased from 0.9 to 5.0. The EDL contraction time decreased from 35 to 31 msec and the indentations increased from 7.3 to 8.1. In spite of the considerable scatter in the data, there was a clear correlation between indentation density and contraction time. Similarly, indentation density was correlated with the voltage dependence of tension generated during K-contractures. The results show that the density of indentations can be modulated by afferent neural activity and support the hypothesis that indentations play an important role in excitation-contraction coupling.
Publisher: Portland Press Ltd.
Date: 04-2004
DOI: 10.1042/BJ20031096
Abstract: We show that peptide fragments of the dihydropyridine receptor II–III loop alter cardiac RyR (ryanodine receptor) channel activity in a cytoplasmic Ca2+-dependent manner. The peptides were AC (Thr-793–Ala-812 of the cardiac dihydropyridine receptor), AS (Thr-671–Leu-690 of the skeletal dihydropyridine receptor), and a modified AS peptide [AS(D-R18)], with an extended helical structure. The peptides added to the cytoplasmic side of channels in lipid bilayers at ≥10 nM activated channels when the cytoplasmic [Ca2+] was 100 nM, but either inhibited or did not affect channel activity when the cytoplasmic [Ca2+] was 10 or 100 µM. Both activation and inhibition were independent of bilayer potential. Activation by AS, but not by AC or AS(D-R18), was reduced at peptide concentrations & µM in a voltage-dependent manner (at +40 mV). In control experiments, channels were not activated by the scrambled AS sequence (ASS) or skeletal II–III loop peptide (NB). Resting Ca2+ release from cardiac sarcoplasmic reticulum was not altered by peptide AC, but Ca2+-induced Ca2+ release was depressed. Resting and Ca2+-induced Ca2+ release were enhanced by both the native and modified AS peptides. NMR revealed (i) that the structure of peptide AS(D-R18) is not influenced by [Ca2+] and (ii) that peptide AC adopts a helical structure, particularly in the region containing positively charged residues. This is the first report of specific functional interactions between dihydropyridine receptor A region peptides and cardiac RyR ion channels in lipid bilayers.
Publisher: Wiley
Date: 07-1991
DOI: 10.1113/JPHYSIOL.1991.SP018684
Abstract: 1. Potassium (K+) contractures have been used to characterize the processes of activation and inactivation of excitation-contraction coupling during prolonged depolarization of fibres in small bundles dissected from rat soleus muscles at 23 degrees C. 2. The smallest measurable K+ contracture tension was recorded with depolarization to -40 mV in 30 mM-K+ and maximum tension was achieved between -26 mV in 80 mM-K+ and -19 mV in 120 mM-K+. 3. The rate of inactivation of K+ contracture tension was voltage dependent. Tension decayed from 80 to 20% of the peak litude within 44.0 +/- 2.2 s at -26 mV (in 80 mM-K+), compared with 66.7 +/- 4.8 s at -35 mV (in 40 mM-K+). Results are given as mean +/- 1 S.E.M. 4. The effect of inactivation on maximum tension was determined using a two pulse protocol in which a 'conditioning' depolarization in solutions containing 20-120 mM-K+ was applied for 0.5-10 min before a 'test' depolarization to -8 mV in 200 mM-K+. The litude of the test contracture was compared with the mean litude of 'control' 200 mM-K+ contractures elicited in normally polarized fibres immediately before and after the two pulse protocol. Conditioning depolarization to -47 mV (in 20 mM-K+) did not reduce test 200 mM-K+ contracture tension. Significant inactivation was seen with further conditioning depolarization to more positive potentials: after 10 min at -40 mV (in 30 mM-K+), or -35 mV (in 40 mM-K+), test 200 mM-K+ contracture tension was reduced by 33 and 70% respectively. 5. In contrast to hibian muscle, where maximum tension falls to zero within a few minutes of depolarization to potentials positive to -50 mV, test 200 mM-K+ contracture tension in rat soleus fibres fell initially rapidly and then slowly, but was not reduced to zero, even after 10 min at -19 mV in 120 mM-K+. 6. The fast phase of inactivation of test 200 mM-K+ contracture tension occurred during the decay of the conditioning K+ contracture. The slow phase of inactivation reached completion after 10 min of conditioning depolarization and occurred during the period when conditioning tension was reduced to zero or to a plateau level. Both phases of inactivation in rat soleus fibres are slow compared with fast and slow inactivation times of 5-100 s respectively reported for hibian muscle. 7. When repolarized after prolonged depolarization, the muscle fibres were initially refractory, i.e. unable to produce tension in response to electrical stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)
Publisher: Oxford University Press (OUP)
Date: 22-06-2005
DOI: 10.1093/HMG/DDI223
Abstract: Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder caused by a CTG repeat expansion in the DMPK gene. Aberrant splicing of several genes has been reported to contribute to some symptoms of DM1, but the cause of muscle weakness in DM1 and elevated Ca2+ concentrations in cultured DM muscle cells is unknown. Here, we investigated the alternative splicing of mRNAs of two major proteins of the sarcoplasmic reticulum, the ryanodine receptor 1 (RyR1) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) 1 or 2. The fetal variants, ASI(-) of RyR1 which lacks residue 3481-3485, and SERCA1b which differs at the C-terminal were significantly increased in skeletal muscles from DM1 patients and the transgenic mouse model of DM1 (HSA(LR)). In addition, a novel variant of SERCA2 was significantly decreased in DM1 patients. The total amount of mRNA for RyR1, SERCA1 and SERCA2 in DM1 and the expression levels of their proteins in HSA(LR) mice were not significantly different. However, heterologous expression of ASI(-) in cultured cells showed decreased affinity for [3H]ryanodine but similar Ca2+ dependency, and decreased channel activity in single-channel recording when compared with wild-type (WT) RyR1. In support of this, RyR1-knockout myotubes expressing ASI(-) exhibited a decreased incidence of Ca2+ oscillations during caffeine exposure compared with that observed for myotubes expressing WT-RyR1. We suggest that aberrant splicing of RyR1 and SERCA1 mRNAs might contribute to impaired Ca2+ homeostasis in DM1 muscle.
Publisher: Elsevier BV
Date: 08-2006
Publisher: Public Library of Science (PLoS)
Date: 22-03-2013
Publisher: Elsevier BV
Date: 08-2018
DOI: 10.1016/J.BIOCEL.2018.05.004
Abstract: Calcium release from internal stores is a quintessential event in excitation-contraction coupling in cardiac and skeletal muscle. The ryanodine receptor Ca
Publisher: Wiley
Date: 05-1973
DOI: 10.1113/JPHYSIOL.1973.SP010208
Abstract: 1. The area and circumference of surface fibres of sartorius muscles were measured from photomicrographs of frozen sections of whole muscles, and compared with the values obtained assuming a circular cross-section. The latter assumption gave an over-estimate of the mean area of 28%, but only a 2% over-estimate of the circumference. In isolated, single fibres, the assumption gave over-estimates of 25 and 6%, of area and circumference respectively.2. The passive electrical properties of fibres were different in summer and winter. The mean internal resistivity, membrane resistance and membrane capacitance were 147 Omega.cm, 7.6 kOmega.cm(2) and 4 muF/cm(2) in summer, and 194 Omega.cm, 3.9 kOmega.cm(2) and 6.7 muF/cm(2) in winter, in fibres of comparable diameters in situ. In single fibres in summer, the mean values were 120 Omega.cm, 8.6 kOmega.cm(2) and 3.6 muF/cm(2).3. In glycerol-treated fibres the mean specific membrane capacitance was 1.0 muF/cm(2) in summer and 2.0 muF/cm(2) in winter. The internal resistivity and specific membrane resistance were 167 Omega.cm and 8.9 kOmega.cm(2) in summer, and 232 Omega.cm and 3.9 kOmega.cm(2) in winter.4. Early after-depolarizations were recorded in glycerol-treated fibres which had a low membrane capacitance, did not twitch and showed little creep'. Electron micrographs of glycerol-treated fibres showed disruption of the transverse tubular system and sarcoplasmic reticulum.5. After exposure of muscles to 400 mM urea or acetamide for 1 hr, muscle fibres did not twitch and had a reduced membrane capacitance in Ringer solution.
Publisher: The Royal Society
Date: 22-01-1992
Abstract: The distribution of ryanodine receptor calcium-release channels over the terminal cisternae (TC) membrane of skeletal muscle fibres was examined by using immunogold electron microscopy. Two monoclonal antibodies (5C3 and 8E2) that bound to monomers of the ryanodine receptor protein on Western blots of SDS-polyacrylamide gels were used to locate calcium-release channels in longitudinal sections of rat sternomastoid and diaphragm fibres. Up to 21% of 5C3 binding on TC membranes was extra-junctional, compared with 46% for 8E2. Binding of 8E2 to the fibres was less than half that of 5C3, possibly because of steric shielding of the 8E2 antigenic site at the junction. The distances between neighbouring particles in clusters was 20-40 nm, i.e. the distance between subunits of the ryanodine receptor or between neighbouring foot structures. We suggest that, during activation, extra-junctional ryanodine receptors may release Ca2+ directly into the myoplasm, rather than into the restricted space of the triad junction.
Publisher: Springer Science and Business Media LLC
Date: 03-1977
DOI: 10.1038/266075A0
Publisher: Springer Science and Business Media LLC
Date: 05-2005
Publisher: Elsevier BV
Date: 10-1991
DOI: 10.1016/0304-3940(91)90610-6
Abstract: The density of Mg(2+)-dependent Ca2+ ATPase in the terminal cisternae of pig skeletal muscle fibers was investigated to discover whether a reduction in Ca2+ ATPase content impairs Ca2+ sequestration and contributes to the elevated myoplasmic Ca2+ concentration in malignant hyperthermia. Unexpectedly, immunogold electron microscopy showed an increase in Ca2+ ATPase, while densitometry of SDS-polyacrylamide gels suggested that the Ca2+ ATPase content of terminal cisternae vesicles did not change. The affinity of Ca2+ ATPase in vesicles for our monoclonal antibody was not altered. We suggest that the availability of antigenic sites in malignant hyperthermia increases after processing for electron microscopy, perhaps as a consequence of altered sarcoplasmic reticulum membrane properties.
Publisher: Wiley
Date: 1999
DOI: 10.1111/J.1469-7793.1999.313AE.X
Abstract: 1. Ryanodine receptor (RyR) Ca2+ channels in the sarcoplasmic reticulum (SR) of skeletal muscle are regulated by the 12 kDa FK506- (or rapamycin-) binding protein (FKBP12). Rapamycin can also activate RyR channels with FKBP12 removed, suggesting that compounds with macrocyclic lactone ring structures can directly activate RyRs. Here we tested this hypothesis using two other macrocyclic lactone compounds, ivermectin and midecamycin. 2. Rabbit skeletal RyRs were examined in lipid bilayers. Ivermectin (cis, 0.66-40 microM) activated six of eight native, four of four control-incubated and eleven of eleven FKBP12-'stripped' RyR channels. Midecamycin (cis, 10-30 microM) activated three of four single native channels, six of eight control-incubated channels and six of seven FKBP12-stripped channels. Activity declined when either drug was washed out. 3. Neither ivermectin nor midecamycin removed FKBP12 from RyRs. Western blots of terminal cisternae (TC), incubated for 15 min at 37 C with 40 microM ivermectin or midecamycin, showed normal amounts of FKBP12. In contrast, no FKBP12 was detected after incubation with 40 microM rapamycin. 4. Ivermectin reduced Ca2+ uptake by the SR Ca2+-Mg2+-ATPase. Ca2+ uptake by TC fell to approximately 40% in the presence of ivermectin (10 microM), both with and without 10 microM Ruthenium Red. Ca2+ uptake by longitudinal SR also fell to approximately 40% with 10 microM ivermectin. Midecamycin (10 microM) reduced Ca2+ uptake by TC vesicles to approximately 76% without Ruthenium Red and to approximately 90 % with Ruthenium Red. 5. The rate of rise of extravesicular [Ca2+] increased approximately 2-fold when 10 microM ivermectin was added to TC vesicles that had been partially loaded with Ca2+ and then Ca2+ uptake blocked by 200 nM thapsigargin. Ivermectin also potentiated caffeine-induced Ca2+ release to approximately 140% of control. These increases in Ca2+ release were not seen with midecamycin. 6. Ivermectin, but not midecamycin, reversibly reduced Ca2+ loading in four of six skinned rat extensor digitorum longus (EDL) fibres to approximately 90%, and reversibly increased submaximal caffeine-induced contraction in five of eight fibres by approximately 110% of control. Neither ivermectin nor midecamycin altered twitch or tetanic tension in intact EDL muscle fibres within 20 min of drug addition. 7. The results confirm the hypothesis that compounds with a macrocyclic lactone ring structure can directly activate RyRs. Unexpectedly, ivermectin also reduced Ca2+ uptake into the SR. These effects of ivermectin on SR Ca2+ handling may explain some effects of the macrolide drugs on mammals.
Publisher: Elsevier BV
Date: 07-1999
Publisher: Wiley
Date: 27-12-2016
Abstract: The core skeletal muscle ryanodine receptor (RyR1) calcium release complex extends through three compartments of the muscle fibre, linking the extracellular environment through the cytoplasmic junctional gap to the lumen of the internal sarcoplasmic reticulum (SR) calcium store. The protein complex is essential for skeletal excitation-contraction (EC)-coupling and skeletal muscle function. Its importance is highlighted by perinatal death if any one of the EC-coupling components are missing and by myopathies associated with mutation of any of the proteins. The proteins essential for EC-coupling include the DHPR α
Publisher: Springer Science and Business Media LLC
Date: 09-1995
DOI: 10.1007/BF00235394
Publisher: Elsevier BV
Date: 11-2009
Publisher: Springer Science and Business Media LLC
Date: 09-1979
DOI: 10.1007/BF01869290
Publisher: Wiley
Date: 03-1992
DOI: 10.1113/JPHYSIOL.1992.SP019031
Abstract: 1. The effects of perchlorate (ClO4-) on contraction have been studied in rat soleus muscle fibres using (i) potassium (K+) contracture and (ii) two-microelectrode-point voltage cl techniques. 2. Membrane potentials (Vm) at all external [K+] were 3-5 mV more negative in ClO4-. The hyperpolarization could not be attributed to a change in Na+, K+, or Cl- permeability, or to an effect on the Na(+)-K+ pump. 3. ClO4- shifts the voltage dependence of tension activation, and contraction threshold, to more negative membrane potentials without altering maximum tension. Consequently, twitches and submaximal K+ contractures were potentiated, whereas tetanic contractions and 200 mM-K+ contractures were unaltered. 4. The decay of K+ contractures during steady depolarization with ClO4- developed a slow exponential phase with an average time constant of 6.05 +/- 0.76 min at -38 mV, and 1.68 +/- 0.15 min at -19 mV. This slow component was (a) under the rapid control of the surface Vm and (b) did not depend on external Ca2+. 5. Inactivation of E-C coupling was measured with a test 200 mM-K+ depolarization following 3-10 min depolarizations in conditioning solutions containing 20-120 mM-K+. ClO4- induced a negative shift in the curve-relating test K+ contracture litude to conditioning Vm but did not alter the rate of repriming of tension upon repolarization. 6. The results suggest that ClO4- increases the amount of activator produced during depolarization and thus allows the slow inactivation step in excitation-contraction (E-C) coupling to be reflected in the decay of K+ contracture tension. 7. A 'perchlorate contracture', which did not depend on the activation of E-C coupling, was observed. The contracture depended on external Ca2+, but not on voltage-dependent Ca2+ channels or Na(+)-Ca2+ exchange.
Publisher: Elsevier BV
Date: 04-2000
Publisher: Elsevier BV
Date: 02-2014
Publisher: Elsevier BV
Date: 02-2011
Publisher: Springer Science and Business Media LLC
Date: 07-1989
DOI: 10.1007/BF01870792
Abstract: Continuous glucose monitoring (CGM) could drive a paradigm shift in diabetes care, but realization of this promise awaits a complementary shift in the way CGM data is used. The most exciting use for CGM is as the input for automated, closed-loop glucose control. Although first generation CGM devices leave much room for improvement, closed-loop control does not have to wait. Algorithms should target blood glucose levels above the normal range for safety in the setting of imperfect CGM measurements. If the mean glucose under closed-loop control is sufficiently close to the chosen target, hemoglobin A1c goals could be met while minimizing risk of hypoglycemia. CGM may also improve the care of intensive care unit patients treated with intensive insulin therapy and the large numbers of diabetic patients in general hospital wards.
Publisher: Wiley
Date: 30-01-2019
DOI: 10.1111/BPH.14562
Publisher: Springer Science and Business Media LLC
Date: 12-05-2009
DOI: 10.1007/S00249-009-0449-6
Abstract: This review focuses on molecular interactions between calsequestrin, triadin, junctin and the ryanodine receptor in the lumen of the sarcoplasmic reticulum. These interactions modulate changes in Ca(2+) release in response to changes in the Ca(2+) load within the sarcoplasmic reticulum store in striated muscle and are of fundamental importance to Ca(2+) homeostasis, since massive adaptive changes occur when expression of the proteins is manipulated, while mutations in calsequestrin lead to functional changes which can be fatal. We find that calsequestrin plays a different role in the heart and skeletal muscle, enhancing Ca(2+) release in the heart, but depressing Ca(2+) release in skeletal muscle. We also find that triadin and junctin exert independent influences on the ryanodine receptor in skeletal muscle where triadin alone modifies excitation-contraction coupling, while junctin alone supports functional interactions between calsequestrin and the ryanodine receptor.
Publisher: SAGE Publications
Date: 07-1994
DOI: 10.1177/42.7.8014481
Publisher: Wiley
Date: 1985
DOI: 10.1113/JPHYSIOL.1985.SP015541
Abstract: K contractures and asymmetrical charge movement were recorded in extensor digitorum longus (e.d.l.) and soleus muscles that had been denervated for 2-68 days. The relationship between maximum tension during a K contracture and membrane potential shifted to more negative potentials in denervated e.d.l. muscles (by -25 mV on average) and to a lesser extent in soleus (by -8 mV on average), and became steeper, more so in e.d.l. than soleus. Apart from an early negative shift of -11 mV in the voltage dependence of tension in e.d.l. muscles during the first week, the other changes in K contractures following denervation occurred progressively during the first 3 weeks and then stabilized. There was a clear difference in charge movement in denervated e.d.l. fibres but little change in denervated soleus fibres, so that the characteristics of charge movement in e.d.l. and soleus became very similar. The maximum amount of charge movement fell from an average normal value of 23 nC/microF to 6 nC/microF in e.d.l. within the first 2 weeks. The voltage sensitivity shifted to more negative potentials (by about -12 mV on average) within the first week. There was no significant change in the slope of the relationship between charge and membrane potential. The effects of denervation on charge movement could only partly explain the changes in K contractures. The only obvious parallels were the early negative shift in the voltage dependence of charge movement and tension in denervated e.d.l. fibres. The other changes in K contractures in denervated fibres could be due to a change in the relationship between charge movement and Ca concentration in the myoplasm or an increase in the Ca affinity of the myofilaments. Although charge movement fell to about a quarter of normal in denervated e.d.l. fibres, membrane capacity increased approximately 3-fold. A similar increase in capacity in soleus fibres was not associated with a change in charge movement. Fewer indentations were seen in denervated than in normal e.d.l. fibres. The decrease paralleled the fall in charge movement.
Publisher: Springer Science and Business Media LLC
Date: 1984
DOI: 10.1007/BF01925971
Publisher: Elsevier BV
Date: 10-1997
DOI: 10.1016/S0006-3495(97)78222-5
Abstract: Widespread inappropriate antibiotic prescribing is a major driver of resistance. Little is known about antifungal prescribing practices in the United States, which is concerning given emerging resistance in fungi, particularly to azole antifungals. We analyzed outpatient U.S. antifungal prescribing data to inform stewardship efforts. Descriptive analysis of outpatient antifungal prescriptions dispensed during 2018 in the IQVIA Xponent database. Prescriptions were summarized by drug, sex, age, geography, and healthcare provider specialty. Census denominators were used to calculate prescribing rates among demographic groups. Healthcare providers prescribed 22.4 million antifungal courses in 2018 (68 prescriptions per 1,000 persons). Fluconazole was the most common drug (75%), followed by terbinafine (11%) and nystatin (10%). Prescription rates were higher among females vs. males (110 vs. 25 per 1,000) and adults vs. children (82 vs. 27 per 1,000). Prescription rates were highest in the South (81 per 1,000 persons) and lowest in the West (48 per 1,000 persons). Nurse practitioners and family practitioners prescribed the most antifungals (43% of all prescriptions), but the highest prescribing rates were among obstetrician-gynecologists (84 per provider). Prescribing of antifungal drugs in the outpatient setting was common, with enough courses dispensed for one in every 15 U.S. residents in 2018. Fluconazole use patterns suggest vulvovaginal candidiasis as a common indication. Regional prescribing differences could reflect inappropriate use or variations in disease burden. Further study of higher antifungal use in the South could help target antifungal stewardship practices.
Publisher: Elsevier BV
Date: 12-1999
Publisher: Elsevier BV
Date: 1996
Publisher: Wiley
Date: 03-10-1994
DOI: 10.1016/0014-5793(94)01001-3
Abstract: 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.
Publisher: Springer Science and Business Media LLC
Date: 06-1987
DOI: 10.1007/BF01871228
Abstract: Skin surface lipid (SSL) film is a mixture of sebum and keratinocyte membrane lipids, protecting skin from environment. Its composition is unique for the high percentage of long chain fatty acids, and of the polyterpenoid squalene, absent in other human tissues, and in non-human Primates sebum. Here, the still incomplete body of information on SSL as mediators of external chemical, physical, and microbial signals and stressors is revised, focusing on the central event of the continuous oxidative modification induced by the metabolic activity of residential and pathological microbial flora, natural or iatrogenic UV irradiation, exposure to chemicals and cosmetics. Once alpha-tocopherol and ubiquinol-10 antioxidant defences of SSL are overcome, oxidation of squalene and cholesterol gives rise to reactive by-products penetrating deeper into skin layers, to mediate local defensive inflammatory, photo-protective, immune reactions or, at higher concentrations, inducing local but also systemic immune depression, ultimately implicating skin cancerogenesis. Qualitative modifications of SSL represent a pathogenetic sign of diagnostic value in dermatological disorders involving altered sebum production, like pytiriasis versicolor, acne, atopic or seborrheic dermatitis, as well as photo-aging. Achievements of nutriceutical interventions aimed at restoring normal SSL composition and homeostasis are discussed, as feasible therapeutic goals and major means of photo-protection.
Publisher: Springer Science and Business Media LLC
Date: 06-10-2015
DOI: 10.1007/S00424-015-1738-9
Abstract: The skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1), essential for excitation-contraction (EC) coupling, demonstrates a known developmentally regulated alternative splicing in the ASI region. We now find unexpectedly that the expression of the splice variants is closely related to fiber type in adult human lower limb muscles. We examined the distribution of myosin heavy chain isoforms and ASI splice variants in gluteus minimus, gluteus medius and vastus medialis from patients aged 45 to 85 years. There was a strong positive correlation between ASI(+)RyR1 and the percentage of type 2 fibers in the muscles (r = 0.725), and a correspondingly strong negative correlation between the percentages of ASI(+)RyR1 and percentage of type 1 fibers. When the type 2 fiber data were separated into type 2X and type 2A, the correlation with ASI(+)RyR1 was stronger in type 2X fibers (r = 0.781) than in type 2A fibers (r = 0.461). There was no significant correlation between age and either fiber-type composition or ASI(+)RyR1/ASI(-)RyR1 ratio. The results suggest that the reduced expression of ASI(-)RyR1 during development may reflect a reduction in type 1 fibers during development. Preferential expression of ASI(-) RyR1, having a higher gain of in Ca(2+) release during EC coupling than ASI(+)RyR1, may compensate for the reduced terminal cisternae volume, fewer junctional contacts and reduced charge movement in type 1 fibers.
Publisher: Wiley
Date: 05-1988
DOI: 10.1113/JPHYSIOL.1988.SP017068
Abstract: 1. Twitches, tetanic contractions and potassium contractures were recorded isometrically from small bundles of rat soleus muscle fibres. 2. Solutions with reduced calcium concentrations (low-calcium solutions), whether buffered with EGTA (85 and 3 microM-Ca2+) or not (15 microM-Ca2+), caused an initial potentiation of contraction followed by depression. 3. The decay of potassium contractures (200 mM-potassium) was more rapid than normal in low-calcium solutions. 4. Recovery from the inactivation produced by a 200 mM-potassium contracture was slowed in low-calcium solutions but full recovery was seen within 10-15 min after return to a solution containing 2.5 mM-Ca2+. 5. Nifedipine (50 microM) in solutions containing 2.5 mM-Ca2+ potentiated contraction whereas, in low-calcium solutions, contraction was depressed and the depression was more pronounced the lower the Ca2+ concentration. 6. As with low-calcium solutions, potassium contractures decayed more rapidly in solutions containing nifedipine. Nifedipine slowed still further the rate of recovery from inactivation in low-calcium solutions. 7. (-) Bay K 8644 (50 microM) depressed contraction, increased the rate of decay of potassium contractures and slowed recovery from inactivation, like nifedipine. The racemate of Bay K 8644 was less effective. 8. In explanation of these and other observations, it is proposed that there is a dihydropyridine-binding molecule in the walls of the transverse tubular system that normally exists predominantly in a 'precursor' form at the resting membrane potential and is converted by membrane depolarization to an 'activator' form essential for excitation-contraction coupling. Conversion of the precursor to activator involves both conformational change and dissociation of calcium. Prolonged depolarization converts activator to an inactivated form by inducing further conformational change and dissociation of calcium. Recovery from inactivation requires reverse conformational changes and rebinding of calcium. The dihydropyridines affect contraction by reducing the affinity of the molecule for calcium.
Publisher: Elsevier BV
Date: 03-2017
DOI: 10.1016/J.YJMCC.2017.01.016
Abstract: Heart failure is a multimodal disorder, of which disrupted Ca To assess Ca The function of RyR2 from left ventricular s les was assessed using lipid bilayer single-channel measurements, whilst RyR2 modification and protein:protein interactions were determined using Western Blots and co-immunoprecipitation. In all failing hearts there was an increase in RyR2 activity at end-diastolic cytoplasmic Ca Despite different aetiologies, disrupted RyR2 Ca
Publisher: Wiley
Date: 07-1982
Abstract: Freeze-fracture replicas of normal and dystrophic C57BL mouse muscle and kidney were examined to see whether here was a deficit in plasmalemmal particles which others suggest is a feature of dystrophies. When compared with normal membranes there was an increase in the particle density in dystrophic extensor digitorum longus muscle, a decrease in dystrophic soleus muscle, and no change in dystrophic kidney. Therefore there was not a general deficit in intramembrane particles in this dystrophic tissue. Indirect evidence supported the hypothesis that abnormalities in dystrophic mouse muscles are caused by abnormal motor input. The density of indentations, parallel to the T-tubule, on the flat surface of the terminal cisternae can be modulated by the motor nerve. Changes were found in indentation density in dystrophic muscle which were similar to changes seen after transection of the spinal cord in the mid-thoracic region. There were parallel changes in contractile properties and indentation density in dystrophic fibers.
Publisher: Wiley
Date: 10-1973
DOI: 10.1113/JPHYSIOL.1973.SP010350
Abstract: Changes in membrane capacity and excitation-contraction coupling caused by glycerol movements have been investigated in toad sartorius fibres using a standard glycerol-Ringer solution containing 400 mM glycerol.1. The rates of glycerol movement, in and out of fibres, were determined by measuring diameter changes in single fibres. Glycerol equilibrated across the surface membrane within 20-25 min after changes in extracellular glycerol concentration.2. The reduction in membrane capacity, which occurs when glycerol-loaded fibres are returned to Ringer solution, was slower than, and not dependent on, changes in fibre volume.3. To obtain the maximum reduction in membrane capacity, it was necessary to expose fibres to glycerol-Ringer for 50-60 min and then to return them to Ringer for at least 60 min. If preparations were not kept in Ringer for the full 50-60 min, the reduction in membrane capacity could be partially or completely reversed by returning the fibres to glycerol-Ringer.4. When fibres were exposed to glycerol-Ringer there was an initial transient contracture: twitches and K contractures were rapidly inhibited, and then slowly recovered over the next 40-50 min. In some preparations, eventual potentiation of twitches was seen.5. When returned to Ringer solution after 60 min in glycerol-Ringer, preparations lost twitches and K contractures within 5-10 min. The time course of this effect was very similar to the time course of the recovery of the normal volume after the initial swelling.6. The briefer the exposure to the glycerol-Ringer, the slower the loss of twitches and K contractures on return to Ringer. In contrast to the loss of membrane capacity, the lesion of excitation-contraction coupling was essentially irreversible. Exposure times as brief as 5 min were eventually effective in producing paralysed fibres which, however, still responded to caffeine.7. The differences in the glycerol load-times required to produce decoupling of excitation and contraction, and disconnexion of the transverse tubules, show that the two effects are separable and that the first does not depend on the second.8. It is proposed that the two lesions obtained in glycerol-treated fibres may be related to distension of the transverse tubular system. It is shown in an Appendix that the outward movement of glycerol from sarcoplasm to transverse tubules would be expected to produce some early swelling of the tubules and this is related to the loss of contraction. Furthermore, much greater swelling would occur if a slow-loading compartment (presumed to be the sarcoplasmic reticulum) dumped glycerol into the transverse tubules: it is thought that this is related to the disconnexion of the transverse tubules.
Publisher: Elsevier
Date: 2017
DOI: 10.1016/BS.APHA.2016.12.001
Abstract: Ryanodine receptor (RyR) ion channels are essential for skeletal and cardiac muscle function. Their knockout leads to perinatal death from respiratory and cardiac failure. Acquired changes or mutations in the protein cause debilitating skeletal myopathy and cardiac arrhythmia which can be deadly. Knowledge of the pharmacology of RyR channels is central to developing effective and specific treatments of these myopathies. The ion channel is a >2.2MDa homotetamer with distinct structural and functional characteristics giving rise to a myriad of regulatory sites that are potential therapeutic targets. Australian researchers have been intimately involved in the exploration of the proteins since their identification in the mid-1980s. We discuss major aspects of RyR physiology and pharmacology that have been tackled in Australian laboratories. Specific areas of interest include ultrastructural aspects and mechanisms of RyR activation in excitation-contraction (EC) coupling and related pharmacological developments, regulation of RyRs by alent cations, by associated proteins including the FK506-binding proteins, by redox factors and phosphorylation. We consider adverse effects of anthracycline chemotherapeutic drugs on cardiac RyRs. Phenotypes associated with RyR mutations are discussed with current and developing therapeutic approaches for treating the underlying RyR dysfunction.
Publisher: Springer Science and Business Media LLC
Date: 06-1986
DOI: 10.1007/BF01753555
Abstract: The objective of this research was to assess the physical activity levels among a unique cohort of Western Australian (WA) mothers with young children who attend a WA Playgroup. Associated factors were also investigated, including self-efficacy for physical activity, social support for exercise, relevant socio-demographic correlates, as well as the stages of change construct within the Transtheoretical Model (TTM). 421 women completed a questionnaire assessing physical activity behaviours. Of these, 368 participants completed the relevant physical activity evaluation items. 82.5% and 17.5% of the s le were classified as active and inactive, respectively. Associations between physical activity status and exercise stage of change were found. Additional associations were established for partner support and self-efficacy for physical activity. The majority of the s le was classified as active. Despite the high percentage of active participants, this study confirms the usefulness of the stages of change measure in that it can be utilised by health promotion practitioners to report physical activity behaviour and develop appropriate intervention strategies among a time poor and hard to reach population. Specifically the results are relevant to mothers in over 16,000 WA families who are involved with Playgroup WA programs. Interventions aimed at improving physical activity levels in mothers with young children should also consider the need to improve self-efficacy and social support.
Publisher: Elsevier BV
Date: 03-2004
Publisher: Elsevier BV
Date: 02-2006
Publisher: Wiley
Date: 08-1996
DOI: 10.1002/(SICI)1097-4598(199608)19:8<1025::AID-MUS10>3.0.CO;2-W
Publisher: Elsevier BV
Date: 12-1981
DOI: 10.1016/0304-3940(81)90443-2
Abstract: There are fewer indentations on the flat surfaces of terminal cisternae in soleus (slow-twitch) than in extensor digitorum longus (EDL, fast-twitch) muscle fibres of rats. Following mid-thoracic spinal cord transection, there is an increase in the number of indentations in soleus fibres but no change in EDL fibres. The increase in the numbers of indentations after spinal cord transections is correlated with changes in the contractile and charge movement properties of the soleus fibres so that they resemble normal EDL fibres. The indentations appear to have an important role in excitation-contraction coupling.
Publisher: Springer Science and Business Media LLC
Date: 30-10-1999
Abstract: Cd(2+)-induced contractures began with a delay of approximately =4 min after adding 3 mM Cd2+ to external solutions that contained Cl- as the major anion. Tension increased to approximately =20% of peak tetanic tension after 30 min and was maintained after Cd2+ washout. Tension developed more rapidly at higher [Cd2+] (up to 10 mM). There was a lack of correlation between the delay before the contracture and contracture tension: (1) tension was reduced by 2 mM CO2+ or 50 microM nifedipine, although the delay remained at approximately =4 min, and (2) the delay fell to seconds when Cd2+ was added in SO42- solutions, although tension was the same as in Cl- solutions. Since (SO4)2- solutions swell T-tubules, Cd2+ may enter the T-system before inducing contractures. Cd(2+)-induced contractures depended on external [Ca2+] since they were reduced when Ca2+ was omitted from solutions. The contractures did not depend on activation of excitation-contraction coupling, since tension was not altered when the voltage sensor was inactivated by depolarization in 40 mM K+. A small contracture developed with 3 mM Zn2+, but not 3 mM Co2+ or La3+. Both Cd2+ and Zn2+ activated the contractile proteins in skinned fibres. Cd(2+)-induced contractures may depend on external Cd2+ releasing Ca2+ from the sarcoplasmic reticulum (SR), or on Cd2+ entering the fibre, releasing Ca2+ from the SR and/or directly activating the contractile proteins.
Publisher: The Company of Biologists
Date: 2019
DOI: 10.1242/JCS.229039
Abstract: Mutations in the cardiac ryanodine receptor calcium release channel (RyR2) can cause deadly ventricular arrhythmias and atrial fibrillation (AF). The RyR2-P2328S mutation produces catecholaminergic polymorphic ventricular tachycardia (CPVT) and AF in hearts from RyR2P2328S/P2328S (RyR2S/S) mice. We have now examined P2328S RyR2 channels from RyR2S/S hearts. The activity of wild type (WT) and P2328S RyR2 channels was similar at a cytoplasmic [Ca2+] of 1 mM, but P2328S RyR2 was significantly more active than WT at a cytoplasmic [Ca2+] of 1 µM. This was associated with a & -fold shift in the AC50 for Ca2+-activation from ∼3.5 µM Ca2+ in WT RyR2 to ∼320 nM in P2328S channels and an unexpected & -fold shift in the IC50 for inactivation from ∼50 mM in WT channels to ≤7 µM in P2328S channels, into systolic [Ca2+] levels. Unexpectedly, the shift in Ca2+-activation was not associated with changes in subconductance activity, S2806 or S2814 phosphorylation, or FKBP12 bound to the channels. The changes in channel activity with the P2328S mutation correlate with altered Ca2+ homeostasis in myocytes from RyR2S/S mice and the CPVT and AF phenotypes.
Publisher: Rockefeller University Press
Date: 05-1988
DOI: 10.1085/JGP.91.5.737
Abstract: K contractures and two-microelectrode voltage-cl techniques were used to measure inactivation of excitation-contraction coupling in small bundles of fibers from rat extensor digitorum longus (e.d.l.) and soleus muscles at 21 degrees C. The rate of spontaneous relaxation was faster in e.d.l. fibers: the time for 120 mM K contractures to decay to 50% of maximum tension was 9.8 +/- 0.5 s (mean +/- SEM) in e.d.l. and 16.8 +/- 1.7 s in soleus. The rate of decay depended on membrane potential: in e.d.l., the 50% decay time was 14.3 +/- 0.7 s for contractures in 80 mM K (Vm = 25 mV) and 4.9 +/- 0.4 s in 160 mM K (Vm = -3 mV). In contrast to activation, which occurred with less depolarization in soleus fibers, steady state inactivation required more depolarization: after 3 min at -40 mV in 40 mM K, the 200 mM K contracture litude in e.d.l. fell to 28 +/- 10% (n = 5) of control, but remained at 85 +/- 2% (n = 6) of control in soleus. These different inactivation properties in e.d.l. and soleus fibers were not influenced by the fact that the 200 mM K solution used to test for steady state inactivation produced contractures that were maximal in soleus fibers but submaximal in e.d.l.: a relatively similar depression was recorded in maximal (200 mM K) and submaximal (60 and 80 mM K) contracture tension. A steady state "pedestal" of tension was observed with maintained depolarization after K contracture relaxation and was larger in soleus than in e.d.l. fibers. The pedestal tension was attributed to the overlap between the activation and inactivation curves for tension vs. membrane potential, which was greater in soleus than in e.d.l. fibers. The K contracture results were confirmed with the two-microelectrode voltage cl : the contraction threshold increased to more positive potentials at holding potentials of -50 mV in e.d.l. or -40 mV in soleus. At holding potentials of -30 mV in e.d.l. or 0 mV in soleus, contraction could not be evoked by 15-ms pulses to +20 mV. Both K contracture and voltage-cl experiments revealed that activation in soleus fibers occurred with a smaller transient depolarization and was maintained with greater steady state depolarization than in e.d.l. fibers. The K contracture and voltage-cl results are described by a model in which contraction depends on the formation of a threshold concentration of activator from a voltage-sensitive molecule that can exist in the precursor, activator, or inactive states.
Publisher: Elsevier BV
Date: 07-1984
DOI: 10.1016/S0022-5320(84)90179-5
Abstract: The effects of denervation on the structure of the triad and longitudinal sarcoplasmic reticulum have been investigated in freeze-fracture replicas of extensor digitorum longus (EDL) and soleus fibers that had been denervated for 2 to 70 days. In EDL fibers the density of indentations along 1 micron of terminal cisternae fell during the first 2 weeks after nerve section, from a normal value of 7.3 +/- 0.2 (mean +/- 1 SEM) to an average value of 2.0 +/- 0.5 in fibers denervated for 16 to 17 days. Denervation did not change the numbers of indentations in soleus fibers. There was a significant change in the orientation of the triads and organization of the longitudinal sarcoplasmic reticulum in denervated EDL and soleus fibers. The effect of denervation on the density of indentations was best correlated with the effect on asymmetric charge movement, when indentation density was compared with twitch contraction time, the voltage sensitivity of tension, and charge movement. The close relationship between indentation density and charge movement provides compelling evidence for a functional link between the two during excitation-contraction coupling.
Publisher: Wiley
Date: 03-1996
Publisher: Wiley
Date: 08-1995
Abstract: High-frequency fatigue (HFF), the decline of force during continuous tetanic stimulation (lasting 4-40 s), was studied in isolated bundles of rat skeletal muscle fibers. HFF was slower in slow-twitch soleus fibers than in fast-twitch red or white sternomastoid fibers denervation accelerated fatigue in soleus. Maximal 200-mmol/L potassium contractures of normal litude were induced in fatigued fibers, suggesting that crossbridge cycling and the voltage activation of excitation-contraction coupling could still occur maximally, but that activation by action potentials was impaired. An increase in [Na+]o slowed HFF, while a small increase in [K+]o or reduction in [Cl(-)]o accelerated HFF. Increasing the tetanic stimulation frequency exacerbated fatigue. Recovery from HFF proceeded rapidly since force increased markedly within a few seconds when stimulation ceased. These results support the hypothesis that a redistribution of Na+, K+, and Cl- across the transverse tubular membranes during repeated action potential activity induces fatigue by reducing the litude and conduction of action potentials.
Publisher: Wiley
Date: 09-1975
DOI: 10.1113/JPHYSIOL.1975.SP011068
Abstract: The plasmalemmal area of striated muscle fibres is greater than the apparent surface area (A = circumference x length) because of variable folds and the invaginations of the caveolae and T-tubules. Freeze-fracture replicas of the surface membrane of sartorius and semitendinosus muscles from Rana pipiens have been used to determine the numbers and distribution of folds and caveolae at different sarcomere lengths. (1) The plasmalemma folds are variable in size and shape, but are always oriented perpendicular to the long axis of the fibre. The folds vary with stretch, being more prominent at short sarcomere lengths. The caveolae are elliptical invaginations of the plasmalemma which open to the outside by a narrow "neck" of approximately 20 nm. The caveolar lumen has an average long dimension of 81.6 +/- 11.7 nm and an average short dimension of 66.9 +/- 7.9 nm. The caveolar "necks" only can be seen in freeze-fracture replicas and these are distributed in two circumferential bands on either side of the Z-line, and in longitudinal bands separated by distances of 1-5 mum. In the sartorius muscle, at a sarcomere length of 2.8 mum, there is an average number of thirty-seven caveolae per square micrometer of fibre surface. (2) During passive stretch the opening of folds provides membrane for the necessary increase in surface area up to a sarcomere length of about 3.0 mum. This length is defined as the critical sarcomere length (Sc). The number of caveolae remains constant at all sarcomere lengths less than Sc and thus their "necks" have been used as membrane markers to determine the amount of folding at different sarcomere lengths. The membrane area contained in folds and caveolae is expressed as a fraction of the apparent surface area (A). For ex le, in the sartorius muscle, at a sarcomere length of 2.4 mum, the membrane area, excluding the T-tubules, is: A + 0.1A (folding) + 0.7A (caveolae) = 1.8A. (3) For stretch beyond Sc membrane is provided by the opening of caveolae. At a sarcomere length of about 8 mum all the caveolae are open and the fibres rupture with further stretch. (4) The relative contributions of folds and caveolae vary with sarcomere length in a way that is consistent with assumptions of constant volume and plasmalemma area. The maintenance of constant plasmalemma area, even after excessive stretch, suggests that the plasmalemma is relatively inelastic in this situation.
Publisher: Rockefeller University Press
Date: 17-06-2022
Abstract: Flecainide, a cardiac class 1C blocker of the surface membrane sodium channel (NaV1.5), has also been reported to reduce cardiac ryanodine receptor (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ release. It has been introduced as a clinical antiarrhythmic agent for catecholaminergic polymorphic ventricular tachycardia (CPVT), a condition most commonly associated with gain-of-function RyR2 mutations. Current debate concerns both cellular mechanisms of its antiarrhythmic action and molecular mechanisms of its RyR2 actions. At the cellular level, it targets NaV1.5, RyR2, Na+/Ca2+ exchange (NCX), and additional proteins involved in excitation–contraction (EC) coupling and potentially contribute to the CPVT phenotype. This Viewpoint primarily addresses the various direct molecular actions of flecainide on isolated RyR2 channels in artificial lipid bilayers. Such studies demonstrate different, multifarious, flecainide binding sites on RyR2, with voltage-dependent binding in the channel pore or voltage-independent binding at distant peripheral sites. In contrast to its single NaV1.5 pore binding site, flecainide may bind to at least four separate inhibitory sites on RyR2 and one activation site. None of these binding sites have been specifically located in the linear RyR2 sequence or high-resolution structure. Furthermore, it is not clear which of the inhibitory sites contribute to flecainide’s reduction of spontaneous Ca2+ release in cellular studies. A confounding observation is that flecainide binding to voltage-dependent inhibition sites reduces cation fluxes in a direction opposite to physiological Ca2+ flow from SR lumen to cytosol. This may suggest that, rather than directly blocking Ca2+ efflux, flecainide can reduce Ca2+ efflux by blocking counter currents through the pore which otherwise limit SR membrane potential change during systolic Ca2+ efflux. In summary, the antiarrhythmic effects of flecainide in CPVT seem to involve multiple components of EC coupling and multiple actions on RyR2. Their clarification may identify novel specific drug targets and facilitate flecainide’s clinical utilization in CPVT.
Publisher: Springer Science and Business Media LLC
Date: 1999
Abstract: The actions of external Cd2+ on the twitch and tetanic contractions, action potentials and potassium (K+) contractures of rat soleus muscle fibre bundles have been investigated. Cd2+ at 1-1.5 mM did not significantly alter tetanic tension, but increased twitch tension and increased the duration and overshoot of action potentials. At >/=3 mM, Cd2+ (1) depressed tetanic contractions and initially potentiated but later depressed twitches, (2) abolished the action potential overshoot, and (3) shifted peak K+ contracture tension to more positive membrane potentials. Twitch and tetanic contractions, and action potentials remained depressed when Cd2+ was washed out of the bath. The effects of Cd2+ on the twitch, tetanus and action potential were mimicked by Zn2+, while La3+ and Co2+ at 3 mM - or Mg2+ and Ca2+ at 30 mM - depressed peak twitch and tetanic tension, but did not potentiate twitches. The results suggest that: (1) Cd2+ and Zn2+ potentiate twitch tension by prolonging action potential depolarisation (2) Cd2+ depresses twitch and tetanic tension by reducing the action potential overshoot, and causing a positive shift in the voltage dependence of contraction and (3) the irreversible depression of action potential litude in rat soleus muscle is a specific property of Cd2+ and Zn2+ that is not shared by Co2+, Mg2+ or Ca2+.
Publisher: Wiley
Date: 11-1993
DOI: 10.1111/J.1476-5381.1993.TB13932.X
Abstract: 1. The aim of the experiments was to examined the effects of beta-adrenoceptor activation on twitch and tetanic contractions in fast- and slow-twitch mammalian skeletal muscle fibres. Isometric force was recorded from bundles of intact fibres isolated from the normal and denervated slow-twitch soleus and normal fast-twitch sternomastoid muscles of the rat. 2. Terbutaline (10 microM), a beta 2-adrenoceptor agonist, induced an average 15% potentiation of peak twitch and peak tetanic force in normal soleus fibres and abbreviated twitch and tetanic relaxation. In white- and red-sternomastoid fibres, 10 microM terbutaline potentiated peak twitch force by about 7% and slowed twitch relaxation. 3. The potentiation of twitches and tetani by terbutaline was quantitatively similar in normal and denervated soleus fibres. However, in contrast to the normal soleus, terbutaline slowed twitch relaxation and had no effect on tetanic relaxation in denervated soleus fibres. 4. Adrenaline (10 microM) increased peak tetanic force by about 7% in both normal and denervated soleus fibres. 5. Exposure to (+/-)-propranolol (0.1 microM), a general beta-adrenoceptor blocker, completely abolished the tetanus potentiation by terbutaline. 6. Dibutyryl-cyclic AMP (2 mM) mimicked the effects of 10 microM terbutaline on peak tetanic force and tetanic relaxation in normal and denervated soleus fibres. Dibutyryl-cyclic AMP also potentiated peak twitch force in denervated soleus fibres but only after a brief period of twitch depression: the twitch depression might be due to butyrate. 7. The results suggest that the increase in peak twitch and tetanic force and abbreviation of tetanic relaxation induced by terbutaline depend on the activation of beta-adrenoceptors and a consequent increase in the myoplasmic cyclic AMP concentration.
Publisher: Wiley
Date: 03-2005
Publisher: Springer Science and Business Media LLC
Date: 06-1984
DOI: 10.1007/BF00713111
Abstract: Ubiquitin is a small modifier protein which is usually conjugated to substrate proteins for degradation. In recent years, a number of ubiquitin-like proteins have been identified however, their roles in eukaryotes are largely unknown. Here, we describe a ubiquitin-like protein URM1, and found it plays important roles in the development and infection process of the rice blast fungus,
Publisher: Springer Science and Business Media LLC
Date: 06-1984
DOI: 10.1007/BF00713110
Publisher: Portland Press Ltd.
Date: 05-04-2005
DOI: 10.1042/BJ20040786
Abstract: The aim of the present study was to explore interactions between surface-membrane DHPR (dihydropyridine receptor) Ca2+ channels and RyR (ryanodine receptor) Ca2+ channels in skeletal-muscle sarcoplasmic reticulum. The C region (725Phe-Pro742) of the linker between the 2nd and 3rd repeats (II–III loop) of the α1 subunit of skeletal DHPRs is essential for skeletal excitation–contraction coupling, which requires a physical interaction between the DHPR and RyR and is independent of external Ca2+. Little is known about the regulatory processes that might take place when the two Ca2+ channels interact. Indeed, interactions between C fragments of the DHPR (C peptides) and RyR have different reported effects on Ca2+ release from the sarcoplasmic reticulum and on RyR channels in lipid bilayers. To gain insight into functional interactions between the proteins and to explore different reported effects, we examined the actions of C peptides on RyR1 channels in lipid bilayers with three key RyR regulators, Ca2+, Mg2+ and ATP. We identified four discrete actions: two novel, low-affinity (& μM), rapidly reversible effects (fast inhibition and decreased sensitivity to Mg2+ inhibition) and two slowly reversible effects (high-affinity activation and a slow-onset, low-affinity inhibition). Fast inhibition and high-affinity activation were decreased by ATP. Therefore peptide activation in the presence of ATP and Mg2+, used with Ca2+ release assays, depends on a mechanism different from that seen when Ca2+ is the sole agonist. The relief of Mg2+ inhibition was particularly important since RyR activation during excitation–contraction coupling depends on a similar decrease in Mg2+ inhibition.
Publisher: Elsevier BV
Date: 02-1986
DOI: 10.1016/0889-1605(86)90058-3
Abstract: Freeze-fracture methods were used to study the sarcoplasmic reticulum and surface membranes in muscles from rats after chronic administration of triiodothyronine (150 micrograms/kg daily, for 1 to 20 days). The major effect of the hormone on the sarcoplasmic reticulum was to increase the numbers of indentations in the terminal cisternae in parallel with an increase in the speed of the isometric twitch. The indentations increased from 7.3 +/- 0.2 to 10.6 +/- 0.1 (mean +/- 1 SEM)/micron of terminal cisternae in the fast-twitch extensor digitorum longus (EDL) and from 0.9 +/- 0.1 to 4.4 +/- 0.1/micron in slow-twitch soleus fibers. The increase in indentation density in both types of muscle occurred within 10 days of the commencement of hormone injection. During the same period there was a small reduction in the density of intramembrane particles in the plasmalemma and a significant increase in the number of caveolae, from 14.6 +/- 0.25 to 20.4 +/- 0.3/micron2 in EDL fibers, and from 22.9 +/- 0.3 to 28.6 +/- 0.3/micron2 in soleus. The increase in caveolae density was coincident with an increase in the area of T-tubule membrane. The results provide further evidence that the indentations in the terminal cisternae play a functional role in muscle activation and that the caveolae are the surface openings of transverse tubules.
Publisher: IMR Press
Date: 2005
DOI: 10.2741/1626
Abstract: The actions of the recombinant skeletal dihydropyridine receptor II-III loop (SDCL), and the C region peptide (CS) on native skeletal muscle ryanodine receptor Ca2+ release channel (RyR1) have been examined. Three non conserved residues in the "C" region of the skeletal DHPR II-III loop were replaced by the equivalent cardiac residues in SDCLAFP-PTT (A739P, F741T and P742T) and single substitutions made in SDCLA-P, SDCLF-T and SDCLP-T. Wild type SDCL as well as SDCLF-T and SDCLP-T activated RyR1 in lipid bilayers with high affinity (10 nM to 1 microM). Wild type SDCL at higher concentrations inhibited RyR1. In contrast, SDCLAFP-PTT and SDCLA-P inhibited the channels at >or=10 nM. The inhibitory actions of these two skeletal loop mutants were distinctly different from the cardiac II-III loop (CDCL) which, like the wild-type SDCL, activated channels. In contrast to the full loop, the triple A739P, F741T and P742T mutation in peptide CS converted the peptides' function from skeletal-like to cardiac-like. The in idual A739P mutation, but not F741T or P742T, reduced the functional efficacy of CS. None of the mutations significantly altered the NMR-based secondary structure of the C residues in SDCLAFP-PTT or CS. The CS peptide and its mutants, like the cardiac CC peptide, were all partially alpha helical at low temperatures. The results show that residue A739 is critical for the functional consequences of interactions between RyR1 and either the skeletal II-III loop or CS, but that none of A739, F741 or P742 are critical determinants of the structure of the C region.
Publisher: Springer Science and Business Media LLC
Date: 04-1997
Abstract: The gating of ryanodine receptor calcium release channels (RyRs) depends on myoplasmic Ca2+ and Mg2+ concentrations. RyRs from skeletal and cardiac muscle are activated by microm Ca2+ and inhibited by mm Ca2+ and Mg2+. 45Ca2+ release from skeletal SR vesicles suggests two mechanisms for Mg2+-inhibition (Meissner, Darling & Eveleth, 1986, Biochemistry 25:236-244). The present study investigates the nature of these mechanisms using measurements of single-channel activity from cardiac- and skeletal RyRs incorporated into planar lipid bilayers. Our measurements of Mg2+- and Ca2+-dependent gating kinetics confirm that there are two mechanisms for Mg2+ inhibition (Type I and II inhibition) in skeletal and cardiac RyRs. The mechanisms operate concurrently, are independent and are associated with different parts of the channel protein. Mg2+ reduces Po by competing with Ca2+ for the activation site (Type-I) or binding to more than one, and probably two low affinity inhibition sites which do not discriminate between Ca2+ and Mg2+ (Type-II). The relative contributions of the two inhibition mechanisms to the total Mg2+ effect depend on cytoplasmic [Ca2+] in such a way that Mg2+ inhibition has the properties of Types-I and II inhibition at low and high [Ca2+] respectively. Both mechanisms are equally important when [Ca2+] = 10 microm in cardiac RyRs or 1 microm in skeletal RyRs. We show that Type-I inhibition is not the sole mechanism responsible for Mg2+ inhibition, as is often assumed, and we discuss the physiological implications of this finding.
Publisher: Springer Science and Business Media LLC
Date: 19-03-2009
DOI: 10.1007/S00424-009-0664-0
Abstract: The Homer protein family allows clustering and/or functional modulation of many proteins from different calcium signalling complexes including those formed by the ryanodine receptor (RyR) Ca(2+) release channel in skeletal muscle and the heart. Homer1b/c and the cardiac RyR (RyR2) are strongly expressed in the heart and neurons where their interaction with each other may modulate Ca(2+) signalling. However, functional interactions between Homer1b and RyR2 have been poorly defined. Our preliminary data and similar consensus binding sites for Homer in RyR2 and skeletal RyR (RyR1) proteins, led to the hypothesis that Homer may similarly regulate both RyR isoforms. Single-channel and [(3)H]ryanodine binding data showed that RyR2 and RyR1 activity increased to a maximum with ~50-100 nM Homer1b and fell with Homer1b > 200 nM. Homer1b (50 nM) activated RyR2 and RyR1 at all cytosolic [Ca(2+)] estimated EC(50) value of RyR2 diminished from ~2.8 microM Ca(2+) (control) to ~1.9 microM Ca(2+) in the presence of 50 nM Homer1b. Short Homer1 (lacking the coiled-coil multimerisation domain) and Homer1b similarly modulated RyR2, indicating an action through ligand binding, not mutimerisation. These actions of Homer were generally similar in RyR2 and RyR1. The strong functional interactions suggest that Homer1 is likely to be an endogenous modulator of RyR channels in the heart and neurons as well as in skeletal muscle.
Publisher: Public Library of Science (PLoS)
Date: 05-10-2011
Publisher: Wiley
Date: 12-05-2017
DOI: 10.1111/BPH.13807
Publisher: Elsevier BV
Date: 07-1983
DOI: 10.1016/S0022-5320(83)90084-9
Abstract: Indentations (hillocks and dimples) in the terminal cisternae of mammalian and hibian skeletal muscle fibers were studied using freeze-fracture and serial thin-section techniques. The structures were seen in all muscles and had a regular separation from each other and from the T-tubule. Indentations were smaller than fenestrations and formed concavities in, but not macromolecular pores through, the terminal cisternae. The average numbers of indentations in rat muscles (measured along the length of the terminal cisternae, within 150 nm of the triadic junction) varied from 0.9 per micrometer in soleus fibers to 9.6 per micrometer in posterior cricoarytenoid fibers. The average numbers in hibian sartorius fibers varied from 1.6 to 3.6 per micrometer in muscles from different species. The regular alignment of the indentations along the triad, as well as a close correlation between their numbers and the contractile properties of the muscle, suggest that they function in contractile activation and may represent sites of calcium release from the terminal cisternae.
Publisher: Elsevier BV
Date: 12-2001
Publisher: Elsevier BV
Date: 05-2007
DOI: 10.1016/J.CECA.2006.08.004
Abstract: Members of the glutathione transferase (GST) structural family are novel regulators of cardiac ryanodine receptor (RyR) calcium channels. We present the first detailed report of the effect of endogenous muscle GST on skeletal and cardiac RyRs. An Mu class glutathione transferase is specifically expressed in human muscle. An hGSTM2-2-like protein was isolated from rabbit skeletal muscle and sheep heart, at concentrations of approximately 17-93 microM. When added to the cytoplasmic side of RyRs, hGSTM2-2 and GST isolated from skeletal or cardiac muscle, modified channel activity in an RyR isoform-specific manner. High activity skeletal RyR1 channels were inactivated at positive potentials or activated at negative potentials by hGSTM2-2 (8-30 microM). Inactivation became faster as the positive voltage was increased. Channels recovered from inactivation when the voltage was reversed, but recovery times were significantly slowed in the presence of hGSTM2-2 and muscle GSTs. Low activity RyR1 channels were activated at both potentials. In contrast, hGSTM2-2 and GSTs isolated from muscle (1-30 microM) in the cytoplasmic solution, caused a voltage-independent inhibition of cardiac RyR2 channels. The results suggest that the major GST isoform expressed in muscle regulates Ca2+ signalling in skeletal and cardiac muscle and conserves Ca2+ stores in the sarcoplasmic reticulum.
Publisher: Elsevier BV
Date: 05-1980
DOI: 10.1016/0304-3940(80)90038-5
Abstract: Stimulus strength-duration curves for contraction threshold have been obtained from normal and dystrophic C57BL mouse muscle fibres using two microelectrode voltage cl techniques. The threshold membrane potential for activation in dystrophic soleus fibres was further from the resting membrane potential than in normal fibres and was close to the threshold for extensor digitorum longus fibres. When a redistribution if fibre types in dystrophic soleus muscles is taken into account the dystrophic data fell within the normal range of values. It is apparent that the inability of dystrophic C57BL mice to use their hind limbs in the normal way cannot be attributed to a failure of excitation-contraction coupling.
Publisher: Springer Science and Business Media LLC
Date: 22-07-2015
Publisher: Oxford University Press (OUP)
Date: 19-07-2012
DOI: 10.1093/HMG/DDS292
Publisher: Elsevier BV
Date: 1997
DOI: 10.1016/S0006-3495(97)78654-5
Abstract: To report incidence, clinical presentation, and treatment outcome of full-thickness macular hole (FTMHs) diagnosed post pars plana vitrectomy. We retrospectively reviewed the demographics, best-corrected visual acuity (BCVA), indication for the primary vitrectomy, time to diagnose the secondary FTMH, optical coherence tomographic (OCT) appearance, and treatment outcome of FTMHs, occurring after vitrectomy performed between January 2019 and December 2020. Six of 523 vitrectomized eyes developed FTMHs, an incidence of 1.1%. There were five females and one male, mean age of 56.5 years (range 37-85). The indication for primary vitrectomy was rhegmatogenous retinal detachment (RRD) in three eyes, one eye each for sub internal limiting membrane hemorrhage from a ruptured macroaneurysm, vitreous hemorrhage from polypoidal choroidal vasculopathy (PCV), and pre-insertion of Ahmed glaucoma drainage device (GDD). FTMHs occurred within one week to three months after vitrectomy (time from primary vitrectomy to the identification of the secondary MH was a mean of 1.03 months). Mean BCVA in all six MH eyes was log MAR 0.9 (Snellen: 6/54). Anatomical closure was achieved after one surgery in three eyes, two surgeries in 1 eye, after photodynamic therapy (PDT) in the PCV eye, and one patient declined surgery. The mean BCVA in the four surgically closed MH eyes improved marginally from log MAR 0.82 (Snellen: 6/38) to log MAR 0.72 (Snellen: 6/30), mean follow-up 7.6 months. Post-vitrectomy FTMH is rare, and RRD was the commonest indication for initial vitrectomy. We observed that all secondary MHs were closed successfully using the inverted internal limiting membrane (ILM) flap technique with limited improvement in vision. The visual outcome of these secondary MHs trails behind that of idiopathic MHs.
Publisher: Wiley
Date: 10-1971
DOI: 10.1113/JPHYSIOL.1971.SP009626
Abstract: 1. A lethal, water soluble toxin (Maculotoxin, MTX) with a molecular weight less than 540, can be extracted from the salivary glands of an octopus (Hapalochlaena maculosa).2. MTX blocks action potentials in sartorius muscle fibres of toads without affecting the membrane potential. Delayed rectification is not inhibited by the toxin.3. At low concentrations (10(-6)-10(-5) g/ml.) MTX blocks action potentials only after a certain number have been elicited. The number of action potentials, which can be defined accurately, depends on the concentration of MTX and the concentration of sodium ions in the extracellular solution.4. The toxin has no post-synaptic effect at the neuromuscular junction and it is concluded that it blocks neuromuscular transmission by inhibiting action potentials in motor nerve terminals.
Publisher: Wiley
Date: 11-1987
Abstract: The purpose of the study was to investigate rod-like structures (rods) in the terminal cisternae membrane of freeze fracture replicas of fast- and slow-twitch mammalian muscle. The 9 X 50 nm rods crossed the junctional gap perpendicular to the T-tubule membrane and terminated near indentations. Rods are likely to have a structural basis because (1) grooves were seen in the complimentary membrane leaflet, (2) rods were seen in tissue fixed in 0.5, 5, or 6% glutaraldehyde or 5% acrolein, (3) rods were seen in tissue fractured over a range of temperatures from -40 to -196 degrees C, (4) the number of rods was correlated with the contractile properties of fibers, and (5) the density of rods increased when fibers were depolarized before fixation. The rods are in a unique location that would allow them to participate in excitation-contraction coupling, perhaps by transmitting an electrical signal from the T-tubule membrane to calcium release sites in the terminal cisternae.
Publisher: Wiley
Date: 30-06-2009
Publisher: Elsevier BV
Date: 05-2005
Publisher: Portland Press Ltd.
Date: 03-2003
DOI: 10.1042/BJ20021488
Abstract: An α-helical II—III loop segment of the dihydropyridine receptor activates the ryanodine receptor calcium-release channel. We describe a novel manipulation in which this agonist's activity is increased by modifying its surface structure to resemble that of a toxin molecule. In a unique system, native β-sheet scorpion toxins have been reported to activate skeletal muscle ryanodine receptor calcium channels with high affinity by binding to the same site as the lower-affinity α-helical dihydropyridine receptor segment. We increased the alignment of basic residues in the α-helical peptide to mimic the spatial orientation of active residues in the scorpion toxin, with a consequent 2—20-fold increase in the activity of the α-helical peptide. We hypothesized that, like the native peptide, the modified peptide and the scorpion toxin may bind to a common site. This was supported by (i) similar changes in ryanodine receptor channel gating induced by the native or modified α-helical peptide and the β-sheet toxin, a 10—100-fold reduction in channel closed time, with a ≤2-fold increase in open dwell time and (ii) a failure of the toxin to further activate channels activated by the peptides. These results suggest that erse structural scaffolds can present similar conformational surface properties to target common receptor sites.
Publisher: Portland Press Ltd.
Date: 06-2003
DOI: 10.1042/BJ20021763
Abstract: The actions of peptide C, corresponding to 724Glu–Pro760 of the II–III loop of the skeletal dihydropyridine receptor, on ryanodine receptor (RyR) channels incorporated into lipid bilayers with the native sarcoplasmic reticulum membrane show that the peptide is a high-affinity activator of native skeletal RyRs at cytoplasmic concentrations of 100 nM–10 μM. In addition, we found that peptide C inhibits RyRs in a voltage-independent manner when added for longer times or at higher concentrations (up to 150 μM). Peptide C had a random-coil structure indicating that it briefly assumes a variety of structures, some of which might activate and others which might inhibit RyRs. The results suggest that RyR activation and inhibition by peptide C arise from independent stochastic processes. A rate constant of 7.5×105 s−1·M−1 was obtained for activation and a lower estimate for the rate constant for inhibition of 5.9×103 s−1·M−1. The combined actions of peptide C and peptide A (II–III loop sequence 671Thr–Leu690) showed that peptide C prevented activation but not blockage of RyRs by peptide A. We suggest that the effects of peptide C indicate functional interactions between a part of the dihydropyridine receptor and the RyR. These interactions could reflect either dynamic changes that occur during excitation–contraction coupling or interactions between the proteins at rest.
Publisher: The Company of Biologists
Date: 15-11-2016
DOI: 10.1242/JCS.198705
Publisher: Wiley
Date: 18-04-2023
Abstract: The COVID‐19 pandemic has challenged nations states across the world. They have implemented lockdown and social distancing and with the development of vaccines have gone to great lengths to build herd immunity for their populations. As place managers, local government has played a variety of roles supporting central government edicts related to social distancing and supporting local businesses impacted by lockdowns. The research reported here comparing the role local government has played in Australia, Canada, Italy, and New Zealand shows that they have at different times and for different issues been policy takers from central government, policy shapers, and policy makers adapting national strategies. Local government plays an important complementary role with central governments in both unitary and federal systems of government. The paper contributes to the literature on multi‐level governance, place‐based decision‐making, and disaster and emergency management by offering a framework for analysing municipal roles in crises management both in their relationship with higher layers of government and in their acting as locally placed organisations. Cross‐national study: Australia, Canada, Italy, and New Zealand. Examination of local government responses to COVID‐19 pandemic as policy makers, takers, or shapers. Comparison of federal and unitary states.
Publisher: Elsevier BV
Date: 11-2007
DOI: 10.1016/J.JMB.2007.09.041
Abstract: Chloride intracellular channel (CLIC) proteins possess the remarkable property of being able to convert from a water-soluble state to a membrane channel state. We determined the three-dimensional structure of human CLIC2 in its water-soluble form by X-ray crystallography at 1.8-A resolution from two crystal forms. In contrast to the previously characterized CLIC1 protein, which forms a possibly functionally important disulfide-induced dimer under oxidizing conditions, we show that CLIC2 possesses an intramolecular disulfide and that the protein remains monomeric irrespective of redox conditions. Site-directed mutagenesis studies show that removal of the intramolecular disulfide or introduction of cysteine residues in CLIC2, equivalent to those that form the intramolecular disulfide in CLIC1, does not cause dimer formation under oxidizing conditions. We also show that CLIC2 forms pH-dependent chloride channels in vitro with higher channel activity at low pH levels and that the channels are subject to redox regulation. In both crystal forms, we observed an extended loop region from the C-terminal domain, called the foot loop, inserting itself into an interdomain crevice of a neighboring molecule. The equivalent region in the structurally related glutathione transferase superfamily corresponds to the active site. This so-called foot-in-mouth interaction suggests that CLIC2 might recognize other proteins such as the ryanodine receptor through a similar interaction.
Publisher: Springer Science and Business Media LLC
Date: 1990
DOI: 10.1007/BF00373620
Abstract: Suppression of the primary response of rabbits to intravenously administered KLH can be achieved with very small amounts of hyperimmune anti-KLH administered a day later since the rabbit apparently rapidly eliminates most of the KLH by nonimmunologic means. The amount of passive anti-KLH needed to achieve immunosuppression was directly proportional to the dose of injected antigen. Antibody passively administered as much as 6-8 days after antigen still can be strongly immunosuppressive, which suggests that the antibody must be reacting with immunogen in or on responding cells or perhaps in the process of transfer between cells. There was no evidence that the presence of passively administered hyperimmune anti-KLH prior to the injection of antigen had any immunosuppressive action beyond the direct neutralization of the injected antigen. When KLH was injected in Freund adjuvant, anti-KLH incorporated with the KLH in the adjuvant was much more efficient in causing immunosuppression than anti-KLH given intravenously. The primary responses to 2 mg KLH given intravenously and 2 microg given in adjuvant reached approximately equal peaks and were suppressible by comparable amounts of intravenously administered anti-KLH. Two observations suggest that passive antibody neutralizes the immunogenic stimulus at the level of in idual antigenic determinants and not merely by aggregating or precipitating entire antigenic molecules. First, anti-abalone hemocyanin (AH) which cross-reacts approximately 50% with KLH was only partially immunosuppressive even in extremely large amounts, i.e., amounts which could react with and precipitate much more KLH than could the smaller but more suppressive doses of anti-KLH. Second, when KLH and anti-KLH were given together in adjuvant, effective immunosuppression was achieved only with amounts of anti-KLH sufficient to saturate or cover virtually all available antigenic determinants. The immunosuppressive quality of passive antibody increases with time after immunization and with repeated immunization of the donor. In view of their relatively weak immunosuppressive properties, antibodies formed in the first weeks of a primary response may not contribute significantly to the turning off of the antibody response. In any event, results obtained by passive transfer of hyperimmune antibody to animals early in a primary response cannot be applied to the natural events in a primary response.
Publisher: Rockefeller University Press
Date: 08-1976
DOI: 10.1083/JCB.70.2.419
Abstract: Electrical uncoupling of crayfish septate lateral giant axons is paralleled by structural changes in the gap junctions. The changes are characterized by a tighter aggregation of the intramembrane particles and a decrease in the overall width of the junction and the thickness of the gap. Preliminary measurements indicate also a decrease in particle diameter. The uncoupling is produced by in vitro treatment of crayfish abdominal cords either with a Ca++, Mg++-free solution containing EDTA, followed by return to normal saline (Van Harreveld's solution), or with VAn Harreveld's solution containing dinitrophenol (DNP). The uncoupling is monitored by the intracellular recording of the electrical resistance at a septum between lateral giant axons. The junctions of the same septum are examined in thin sections those of other ganglia of the same chain used for the electrical measurements are studied by freeze-fracture. In controls, most junctions contain a more or less regular array of particles repeating at a center to center distance of approximately 200 A. The overall width of the junctions is approximately 200 A and the gap thickness is 40-50 A. Vesicles (400-700 A in diameter) are closely apposed to the junctional membranes. In uncoupled axons, most junctions contain a hexagonal array of particles repeating at a center to center distance of 150-155 A. The overall width of the junctions is approximately 180 A and the gap thickness is 20-30 A. These junctions are usually curved and are rarely associated with vesicles. Isolated, PTA-stained junctions, also believed to be uncoupled, display similar structural features. There are reasons to believe that the changes in structure and permeability are triggered by an increase in the intracellular free Ca++ concentration. Most likely, the changes in permeability are caused by conformational changes in some components of the intramembrane particles at the gap junctions.
Publisher: American Medical Association (AMA)
Date: 12-2019
Publisher: Elsevier BV
Date: 1994
DOI: 10.1016/0304-3940(94)90471-5
Abstract: Monoclonal antibodies against ryanodine receptor (5C3) and calcium ATPase (D12) of skeletal muscle sarcoplasmic reticulum were used in an immunoelectron microscopic study of cerebellar Purkinje cells and neurons of the hypoglossal and dorsal motor nuclei of the vagus (DMV) from rat and guinea-pig. All neurones were labelled with D12 and all, except rat DMV, labelled with 5C3. Most labelling was on smooth endoplasmic reticulum within 500 nm of the plasmalemma where Ca(2+)-activated Ca2+ release would rapidly increase cytosolic calcium following a small Ca2+ influx across the plasmalemma.
Publisher: Informa UK Limited
Date: 30-03-2011
Publisher: Elsevier BV
Date: 2006
DOI: 10.1016/J.BIOCEL.2006.03.020
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 alpha-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K+ channels. The peptide (0.1-10 microM) inhibited skeletal ryanodine receptor channels when the cytoplasmic Ca2+ concentration was 1 microM but with 10 microM cytoplasmic Ca2+, skeletal ryanodine receptors were activated by < or = 1.0 microM peptide and inhibited by 10 microM 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.
Publisher: Elsevier BV
Date: 03-2014
DOI: 10.1016/J.BIOCEL.2013.12.001
Abstract: There is an overwhelming body of work supporting the idea that excitation-contraction coupling in skeletal muscle depends on a physical interaction between the skeletal muscle isoform of the dihydropyridine receptor L-type Ca(2+) channel and the skeletal isoform of the ryanodine receptor Ca(2+) release channel. A general assumption is that this physical interaction is between "critical" residues that have been identified in the II-III loop of the dihydropyridine receptor alpha subunit and the ryanodine receptor. However, despite extensive searches, the complementary "critical" residues in the ryanodine receptor have not been identified. This raises the possibility that the coupling proceeds either through other subunits of the dihydropyridine receptor and/or other co-proteins within the large RyR1 protein complex. There have been some remarkable advances in recent years in identifying proteins in the RyR complex that impact on the coupling process, and these are considered in this review. A major candidate for a role in the coupling mechanism is the beta subunit of the dihydropyridine receptor, because specific residues in both the beta subunit and ryanodine receptor have been identified that facilitate an interaction between the two proteins and these also impact on excitation-contraction coupling. This role of beta subunit remains to be fully investigated as well as the degree to which it may complement any other direct or indirect voltage-dependent coupling interactions between the DHPR alpha II-III loop and the ryanodine receptor.
Publisher: Portland Press Ltd.
Date: 11-12-2007
DOI: 10.1042/BJ20060686
Abstract: The aim of the present study was to examine residues that are variably spliced in the juvenile and adult isoforms of the skeletal-muscle RyR1 (type 1 ryanodine receptor). The juvenile ASI(−) splice variant is less active than the adult ASI(+) variant and is overexpressed in patients with DM (myotonic dystrophy) [Kimura, Nakamori, Lueck, Pouliquin, Aoike, Fujimura, Dirksen, Takahashi, Dulhunty and Sakoda (2005) Hum. Mol. Genet. 14, 2189–2200]. In the present study, we explore the ASI region using synthetic peptides corresponding to rabbit RyR1 residues Thr3471-Gly3500 either containing [PASI(+)] or lacking [PASI(−)] the ASI residues. Both peptides increased [3H]ryanodine binding to rabbit RyR1s, increased Ca2+ release from sarcoplasmic reti-culum vesicles and increased single RyR1 channel activity. The peptide PASI(−) was more active in each case than PASI(+). [3H]Ryanodine binding to recombinant ASI(+)RyR1 or ASI(−)-RyR1 was enhanced more by PASI(−) than PASI(+), with the greatest increase seen when PASI(−) was added to ASI(−)RyR1. The activation of the RyR channels is consistent with the hypo-thesis that the peptides interrupt an inhibitory inter-domain inter-action and that PASI(−) is more effective at interrupting this interaction than PASI(+). We therefore suggest that the ASI(−) sequence interacts more tightly than the ASI(+) sequence with its binding partner, so that the ASI(−)RyR1 is more strongly inhibited (less active) than the ASI(+)RyR1. Thus the affinity of the binding partners in this inter-domain interaction may deter-mine the activities of the mature and juvenile isoforms of RyR1 and the stronger inhibition in the juvenile isoform may contribute to the myopathy in DM.
Publisher: Elsevier BV
Date: 03-2009
DOI: 10.1016/J.BIOCEL.2008.08.004
Abstract: The II-III loop of the dihydropyridine receptor (DHPR) alpha(1s) subunit is a modulator of the ryanodine receptor (RyR1) Ca(2+) release channel in vitro and is essential for skeletal muscle contraction in vivo. Despite its importance, the structure of this loop has not been reported. We have investigated its structure using a suite of NMR techniques which revealed that the DHPR II-III loop is an intrinsically unstructured protein (IUP) and as such belongs to a burgeoning structural class of functionally important proteins. The loop does not possess a stable tertiary fold: it is highly flexible, with a strong N-terminal helix followed by nascent helical/turn elements and unstructured segments. Its residual structure is loosely globular with the N and C termini in close proximity. The unstructured nature of the II-III loop may allow it to easily modify its interaction with RyR1 following a surface action potential and thus initiate rapid Ca(2+) release and contraction. The in vitro binding partner for the II-III was investigated. The II-III loop interacts with the second of three structurally distinct SPRY domains in RyR1, whose function is unknown. This interaction occurs through two preformed N-terminal alpha-helical regions and a C-terminal hydrophobic element. The A peptide corresponding to the helical N-terminal region is a common probe of RyR function and binds to the same SPRY domain as the full II-III loop. Thus the second SPRY domain is an in vitro binding site for the II-III loop. The possible in vivo role of this region is discussed.
Publisher: Wiley
Date: 12-1993
Abstract: We examined the mechanism(s) which allow terbutaline, a beta 2-adrenergic agonist, to increase isometric force in bundles of normal and denervated rat soleus fibers. Terbutaline (10 mumol/L) potentiated tetanic contractions during exposure to 1 mmol/L ouabain, 10 mumol/L nifedipine, or 0.5 mmol/L iodoacetate. Terbutaline induced equivalent increases in submaximal potassium (K+) contracture and tetanic force: these effects were mimicked by 2 mmol/L dibutyryl-cyclic AMP. Therefore, terbutaline increased force by a cyclic AMP-dependent mechanism other than enhancement of sodium-pump activity, dihydropyridine sensitive Ca2+ currents, glycolysis, or action potentials. Pretreatment with 1 mmol/L caffeine induced submaximal potentiation of peak tetanic force but prevented further potentiation by terbutaline. This suggested that terbutaline did not influence the myofilaments, but acted on the sarcoplasmic reticulum (SR) to increase the myoplasmic Ca2+ concentration and hence force production. We speculate that force is potentiated following beta-adrenoceptor activation by a cyclic AMP-dependent phosphorylation of Ca2+ release channels to facilitate SR calcium release during tetanic stimulation.
Publisher: Canadian Science Publishing
Date: 05-1988
DOI: 10.1139/Y88-088
Abstract: The effects of diazepam on potassium contractures, contraction threshold, and resting tension have been examined in rat soleus muscle fibres. Two actions of the drug were defined that could not be attributed to changes in the resting membrane potential or depolarization in high potassium solutions. The major effect was an increase in the litude of submaximal tension during either twitches or potassium contractures and an increase in resting tension. At 400 μM diazepam, there was (a) a fourfold increase in 40 mM potassium contracture tension, (b) a negative shift of 8 mV in the membrane potential for half maximum tension estimated from the best fit of a Boltzmann-type equation to average potassium contracture data, (c) a negative shift of 8 mV in the threshold for contraction measured under voltage cl conditions, and (d) a contracture of variable litude to a level that was occasionally equivalent to maximum tetanic tension. These potentiating actions of diazepam depended on drag concentration within the range of 100–800 μM. In contrast, the second effect of diazepam, depression of maximum tension by 10–15%, was independent of drug concentration between 100 and 400 μM. The results support the idea that diazepam produces an increase in resting myoplasmic calcium concentrations.
Publisher: Rockefeller University Press
Date: 25-08-2014
Abstract: Regulation of the cardiac ryanodine receptor (RyR2) by intracellular Ca2+ and Mg2+ plays a key role in determining cardiac contraction and rhythmicity, but their role in regulating the human RyR2 remains poorly defined. The Ca2+- and Mg2+-dependent regulation of human RyR2 was recorded in artificial lipid bilayers in the presence of 2 mM ATP and compared with that in two commonly used animal models for RyR2 function (rat and sheep). Human RyR2 displayed cytoplasmic Ca2+ activation (Ka = 4 µM) and inhibition by cytoplasmic Mg2+ (Ki = 10 µM at 100 nM Ca2+) that was similar to RyR2 from rat and sheep obtained under the same experimental conditions. However, in the presence of 0.1 mM Ca2+, RyR2s from human were 3.5-fold less sensitive to cytoplasmic Mg2+ inhibition than those from sheep and rat. The Ka values for luminal Ca2+ activation were similar in the three species (35 µM for human, 12 µM for sheep, and 10 µM for rat). From the relationship between open probability and luminal [Ca2+], the peak open probability for the human RyR2 was approximately the same as that for sheep, and both were ∼10-fold greater than that for rat RyR2. Human RyR2 also showed the same sensitivity to luminal Mg2+ as that from sheep, whereas rat RyR2 was 10-fold more sensitive. In all species, modulation of RyR2 gating by luminal Ca2+ and Mg2+ only occurred when cytoplasmic [Ca2+] was & µM. The activation response of RyR2 to luminal and cytoplasmic Ca2+ was strongly dependent on the Mg2+ concentration. Addition of physiological levels (1 mM) of Mg2+ raised the Ka for cytoplasmic Ca2+ to 30 µM (human and sheep) or 90 µM (rat) and raised the Ka for luminal Ca2+ to ∼1 mM in all species. This is the first report of the regulation by Ca2+ and Mg2+ of native RyR2 receptor activity from healthy human hearts.
Publisher: Elsevier BV
Date: 05-2004
Publisher: Springer Science and Business Media LLC
Date: 04-1985
DOI: 10.1007/BF00713062
Abstract: Mitochondrial DNA (mtDNA) is highly polymorphic at the population level, and specific mtDNA variants affect mitochondrial function. With emerging evidence that mitochondrial mechanisms are central to common human diseases, it is plausible that mtDNA variants contribute to the "missing heritability" of several complex traits. Given the central role of mtDNA genes in oxidative phosphorylation, the same genetic variants would be expected to alter the risk of developing several different disorders, but this has not been shown to date. Here we studied 38,638 in iduals with 11 major diseases, and 17,483 healthy controls. Imputing missing variants from 7,729 complete mitochondrial genomes, we captured 40.41% of European mtDNA variation. We show that mtDNA variants modifying the risk of developing one disease also modify the risk of developing other diseases, thus providing independent replication of a disease association in different case and control cohorts. High-risk alleles were more common than protective alleles, indicating that mtDNA is not at equilibrium in the human population, and that recent mutations interact with nuclear loci to modify the risk of developing multiple common diseases.
Publisher: F1000 Research Ltd
Date: 27-11-2018
DOI: 10.12688/F1000RESEARCH.16434.1
Abstract: The ryanodine receptor calcium release channel is central to cytoplasmic Ca 2+ signalling in skeletal muscle, the heart, and many other tissues, including the central nervous system, lymphocytes, stomach, kidney, adrenal glands, ovaries, testes, thymus, and lungs. The ion channel protein is massive (more than 2.2 MDa) and has a structure that has defied detailed determination until recent developments in cryo-electron microscopy revealed much of its structure at near-atomic resolution. The availability of this high-resolution structure has provided the most significant advances in understanding the function of the ion channel in the past 30 years. We can now visualise the molecular environment of in idual amino acid residues that form binding sites for essential modulators of ion channel function and determine its role in Ca 2+ signalling. Importantly, the structure has revealed the structural environment of the many deletions and point mutations that disrupt Ca 2+ signalling in skeletal and cardiac myopathies and neuropathies. The implications are of vital importance to our understanding of the molecular basis of the ion channel’s function and for the design of therapies to counteract the effects of ryanodine receptor-associated disorders.
Publisher: Public Library of Science (PLoS)
Date: 03-09-2020
Publisher: The Company of Biologists
Date: 2015
DOI: 10.1242/JCS.160689
Abstract: Junctin, a non-catalytic splice variant of the aspartate-β-hydroxylase gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca2+ store where it modifies Ca2+ signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca2+ release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca2+ signalling, muscle dysfunction and cardiac arrhythmia. However, junctin's binding interactions with RyRs are largely unknown and have been assumed to occur only in the SR lumen. We find robust binding of RyRs to full junctin, its luminal and unexpectedly its cytoplasmic domain, each with distinct effects on RyR1 and RyR2 activity. Full junctin in the luminal solution increases channel activity by ∼3-fold. The C-terminal luminal interaction inhibits RyR channel activity by ∼50%. The N-terminal cytoplasmic binding produces a ∼5-fold increase in RyR activity. The cytoplasmic interaction is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including S1–S2 linker of RyR1 and N-terminal junctin binds between RyR1 residues 1078-2156.
Publisher: Springer Science and Business Media LLC
Date: 05-09-2016
DOI: 10.1007/S00424-016-1869-7
Abstract: Triadin isoforms, splice variants of one gene, maintain healthy Ca
Publisher: Elsevier BV
Date: 10-1979
DOI: 10.1016/0304-3940(79)96152-4
Abstract: A two pulse technique was used to study the decay of mechanical activation in TTX poisoned mouse extensor digitorum longus (EDL) and soleus muscle fibres. A two microelectrode point voltage cl technique was used with visual endpoint determination of the threshold potential for activation. The excitatory effect of a brief (0.5-2.0 msec) depolarizing pulse decayed quickly and became inhibitory. In EDL fibres the peak inhibition was 0.5-1.0 msec after the termination of the conditioning pulse and decayed within 3.0 msec. Inhibition was less pronounced in soleus fibres and decayed more slowly. It is suggested that inhibition may be responsible for a refractory period in excitation-contraction coupling.
Publisher: Elsevier BV
Date: 04-2001
Publisher: Elsevier BV
Date: 08-2010
DOI: 10.1016/J.BCP.2010.04.019
Abstract: Ca(2+) release from the sarcoplasmic reticulum through cardiac ryanodine receptors (RyR2) is essential for heart function and is inhibited by the carboxy terminal domain of glutathione transferase M2-2 (GSTM2-C) and derivative fragments containing helix 6. Since a peptide encoding helix 6 alone does not fold into a helix and does not inhibit RyR2 Ca(2+) release, the importance of the structure of helix 6 and its role in stabilizing GSTM2-C was tested by inserting potentially destabilizing mutations into this helical segment. GSTM2-C preparations with D156A or L163A mutations were so insoluble that the protein could not be purified. Proteins with F157A and Y260A substitutions were soluble, but had lost their capacity to inhibit both RyR2 Ca(2+) release from vesicles and RyR2 channels in bilayers. Circular dichroism studies indicated that these mutated proteins retained their helical secondary structure, although changes in their endogenous tryptophan fluorescence indicated that the F157A and Y160A mutations caused changes in their folding. The single channel studies were conducted with 2mM ATP and 10microM Ca(2+) in the cytoplasmic solution, mimicking concentrations in the cytosol of cardiac myocytes. Wild type GSTM2-C inhibited RyR2 only at a potential of +40mV, which may develop during Ca(2+) efflux, but not at -40mV. Together, the results indicate that the structure of helix 6 in the C-terminal fold is critical to the inhibitory action of GSTM2-2 and suggest that therapeutics mimicking this structure may reduce excess Ca(2+) release during diastole, which can lead to fatal arrhythmia.
Publisher: SAGE Publications
Date: 10-1993
Abstract: We evaluated the use of immunogold electron microscopy to study the distribution of calcium ATPase in the sarcoplasmic reticulum membrane of skeletal muscle. We examined (a) 1-nm gold labeling, (b) the effect of gold size on immunolabeling, and (c) the densities of gold particles in areas of maximal labeling in fibers from rat extensor digitorum longus and pig gracilis muscles. The technique allowed unequivocal identification of the calcium ATPase. Gold particles of 1 nm were successfully visualized in unstained or lightly stained sections and the density of labeling was about 20 times greater than with 10-nm gold. The average densities in areas of intense labeling were 2878 +/- 139/microns 2 with 5-nm gold and 4310 +/- 276/microns 2 with 1-nm gold. These numbers are similar to the density of particles in freeze-fracture replicas of sarcoplasmic reticulum. The low density of 10-nm gold suggests that the large gold particles hinder binding of secondary to primary antibodies. The difference between 1- and 5-nm gold is explained by the amounts of gold conjugated to the immunoglobulin. The results suggest that there is a one-to-one relationship between secondary immunoglobulins (1-nm or 5-nm gold conjugates) and oligomeric complexes of calcium ATPase.
Publisher: Mary Ann Liebert Inc
Date: 03-2000
Abstract: RyRs contain 80-100 cysteine residues per subunit, of which approximately 25% are free for covalent modification, while the remainder are either modified or form intraprotein disulfides. Oxidizing and nitrosylating reagents have several effects on single RyR channel activity, which depend on the type of modifying reagent, the isoform of the RyR, and ligands bound to the channel. We present evidence here for four major classes of functional cysteine residues associated with RyR channels, i.e., two classes with free -SH groups that either activate or inhibit channels when covalently modified and two classes, with endogenous modification, that either inhibit or activate. Single-channel characteristics provide evidence for four discrete responses within the first activating class, two responses within the second inhibiting class and two types of response within the third endogenously modified class. All but one of these changes in channel properties depend on residues located on the cytoplasmic or membrane-associated domains of the RyR the remaining response is confined to the luminal domain. If it is assumed that each type of response depends on a separate subclass of cysteine residue and that each subclass contains a minimum of one cysteine per subunit, our results suggest that there are at least nine cysteine residues per subunit with functional connections to the gating mechanism of RyR channels. These cysteine residues may be selectively modified under physiological and pathological conditions to regulate Ca2+ release from the sarcoplasmic reticulum and contraction.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 08-08-2014
Publisher: Rockefeller University Press
Date: 30-07-2012
Publisher: Rockefeller University Press
Date: 07-1989
DOI: 10.1085/JGP.94.1.183
Abstract: Inactivation of excitation-contraction coupling was examined in extensor digitorum longus (EDL) and soleus muscle fibers from rats injected daily with tri-iodothyronine (T3, 150 micrograms/kg) for 10-14 d. Steady-state activation and inactivation curves for contraction were obtained from measurements of peak potassium contracture tension at different surface membrane potentials. The experiments tested the hypothesis that noninactivating tension is a "window" tension caused by the overlap of the activation and inactivation curves. Changes in the litude and voltage dependence of noninactivating tension should be predicted by the changes in the activation and inactivation curves, if noninactivating tension arises from their overlap. After T3 treatment, the area of overlap increased in EDL fibers and decreased in soleus fibers and the overlap region was shifted to more negative potentials in both muscles. Noninactivating tension also appeared at more negative membrane potentials after T3 treatment in both EDL and soleus fibers. The effects of T3 treatment were confirmed with a two microelectrode voltage-cl technique: at the resting membrane potential (-80 mV) contraction in response to a brief test pulse required less than normal depolarization in EDL, but more than normal depolarization in soleus fibers. After T3 treatment, the increase in contraction threshold at depolarized holding potentials (attributed to inactivation) occurred at more depolarized holding potentials in EDL, or less depolarized holding potentials in soleus. The changes in contraction threshold could be accounted for by the effects of T3 on the activation and inactivation curves. In conclusion, (a) T3 appeared to affect the expression of both activation and inactivation characteristics, but the activation effects could not be cleanly distinguished from T3 effects on the sarcoplasmic reticulum and contractile proteins, and (b) the experiments provided evidence for the hypothesis that the noninactivating tension is a steady-state "window" tension.
Publisher: Springer Science and Business Media LLC
Date: 09-1993
DOI: 10.1007/BF00211096
Publisher: Springer Science and Business Media LLC
Date: 10-1980
DOI: 10.1007/BF01868828
Publisher: Wiley
Date: 25-02-2009
DOI: 10.1111/J.1440-1681.2008.05094.X
Abstract: 1. Many biological processes that are governed by intracellular calcium signals rely on intracellular stores, which provide a reliable, controlled release of calcium into the cytoplasm. Calcium release through the ryanodine receptor (RyR), the main ion channel in the sarcoplasmic reticulum (the calcium store in muscle) is the key determinant of muscle force. 2. Calsequestrin, the main calcium buffer in the sarcoplasmic reticulum, provides a pool of calcium for release through the RyR and acts as a luminal calcium sensor for the channel via its interactions with triadin and junctin. Until recently, how calsequestrin communicated the store Ca(2+) load to the RyR remained unknown. 3. Calsequestrin 1 (skeletal calsequestrin) has been shown to both inhibit and activate the skeletal RyR1, dependent on whether it's bound to the RyR1 directly or indirectly via anchoring proteins. 4. The phosphorylation status of calsequestrin 1 is deemed important: it influences the Ca(2+) binding capacity of calsequestrin, the way in which calsequestrin 1 regulates the RyR1 and how calsequestrin 1 interacts with the key anchoring protein junctin. 5. In skeletal muscle, junctin plays a more critical role than triadin in the mechanism that controls Ca(2+) release from the sarcoplasmic reticulum. 6. The close relationship between altered expression and dysfunction of calsequestrin in several skeletal and cardiac disorders highlights the critical role that calsequestrin plays in maintaining Ca(2+) homeostasis and regulation of muscle contraction.
Publisher: Elsevier BV
Date: 04-2025
Publisher: Springer Science and Business Media LLC
Date: 28-04-2009
DOI: 10.1007/S00249-009-0455-8
Abstract: 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.
Publisher: Springer Science and Business Media LLC
Date: 08-1987
DOI: 10.1007/BF01568885
Abstract: The Krebs-Henseleit buffer is the best perfusion solution for isolated mammalian hearts. We hypothesized that a Krebs-Henseleit buffer-based cardioplegic solution might provide better myocardial protection than well-known crystalloid cardioplegic solutions because of its optimal electrolyte and glucose levels, presence of buffer systems, and mild hyperosmolarity. Isolated Langendorff-perfused rat hearts were subjected to either global ischemia without cardioplegia (controls) or cardioplegic arrest for either 60 or 180 min, followed by 120 min of reperfusion. The modified Krebs-Henseleit buffer-based cardioplegic solution (mKHB) and St. Thomas' Hospital solution No. 2 (STH2) were studied. During global ischemia, the temperatures of the heart and the cardioplegic solutions were maintained at either 37°C (60 min of ischemia) or 22°C (moderate hypothermia, 180 min of ischemia). Hemodynamic parameters were registered throughout the experiments. The infarct size was determined through histochemical examination. Cardioplegia with the mKHB solution at moderate hypothermia resulted in a minimal infarct size (5 ± 3%) compared to that in the controls and STH2 solution (35 ± 7% and 19 ± 9%, respectively P < 0.001, for both groups vs. the mKHB group). In contrast to the control and STH2-treated hearts, no ischemic contracture was registered in the mKHB group during the 180-min global ischemia. At normothermia, the infarct sizes were 4 ± 3%, 72 ± 6%, and 70 ± 12% in the mKHB, controls, and STH2 groups, respectively (P < 0.0001). In addition, cardioplegia with mKHB at normothermia prevented ischemic contracture and improved the postischemic functional recovery of the left ventricle (P < 0.001, vs. STH2). The data suggest that the Krebs-Henseleit buffer-based cardioplegic might be superior to the standard crystalloid solution (STH2).
Publisher: IMR Press
Date: 2004
DOI: 10.2741/1443
Abstract: Excitation-contraction coupling in skeletal muscle is thought to depend on a physical interaction between II-III loop of the alpha1 subunit of the skeletal dihydropyridine receptor (DHPR) and ryanodine receptor (RyR). A peptide corresponding to II-III loop residues 671-690 of the skeletal DHPR (peptide A) is a high affinity activator of the RyR when it adopts an alpha-helical structure with critical basic residues aligned along one helical surface (1). Neither the structure of the full length II-III loop, or of sequences longer than 671-690 residues have been determined. Here we describe the structure and function of a 40 amino acid peptide corresponding to residues 671-710 (peptide AB) of the skeletal DHPR alpha1 subunit. This peptide contains the A region with a further 20 residues towards the C-terminus of the II-III loop. We predicted that peptide AB would strongly activate the RyR, because (a) it contains the active A sequence of basic residues and (b) it contains a greater proportion of the II-III loop. The structure of the AB peptide was determined and it was found to consist of two helical regions joined by an unstructured linker region. Surprisingly, although the structure of the A region was maintained, the 40 residue peptide was unable to release Ca2+ from skeletal SR. Strong activity was restored when four negatively charged residues in the C-terminal part of the peptide were replaced by neutral residues. The charge substitution caused minimal changes in the overall structural profile of the peptide and virtually no changes in the A portion of the peptide. The results suggest that the ability of the A region of the skeletal II-III loop to interact with the RyR could depend on the tertiary conformation of the II-III loop, which is thought to change during EC coupling.
Publisher: Wiley
Date: 27-11-2018
DOI: 10.1002/JBM.A.36286
Abstract: Proliferative chondrocytes are critical to realize regeneration of damaged epiphyseal growth plate. However, acquiring autologous replacement cells involves highly invasive procedures and often results in limited cell quantity. Mesenchymal stem cells (MSCs) are a potential source of chondrogenic cells for the treatment of cartilage disorders and injuries. The temporal effect of transforming growth factor beta 3 (TGFβ3) and fibroblast growth factor 2 (FGF2) on the derivation of proliferative chondrocytes from MSCs in three-dimensional agarose was investigated by manipulating the duration of TGFβ3 and FGF2 treatment. The differentiation process was monitored by quantitative reverse transcription polymerase chain reaction (qRT-PCR) as well as nanosensors containing two molecular beacons that target critical biomarkers for proliferative chondrocytes (i.e., collagen type-II messenger ribonucleic acid [mRNA] and Ki67 mRNA). The molecular beacon-based nanosensors were found to be comparable to qRT-PCR in measuring mRNA expression and thus providing a noninvasive mean to screen and monitor culture s les. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 895-904, 2018.
Publisher: The Company of Biologists
Date: 2017
DOI: 10.1242/JCS.204461
Abstract: Ryanodine receptor (RyR) calcium channels are central to striated muscle function and influence signalling in neurones and other cell types. Beneficially low RyR activity and maximum conductance opening may be stabilised when RyRs bind to FK506 binding proteins (FKBPs) and destabilised by FKBP dissociation, with submaximal opening during RyR hyperactivity associated with myopathies and neurological disorders. However this is debated and quantitative evidence is lacking. Here we have measured altered FKBP binding to RyRs and submaximal activity with addition of wild-type (WT) CLIC2, an inhibitory RyR ligand, or its H101Q mutant that hyperactivates RyRs, likely causing cardiac and intellectual abnormalities. The proportion of sub-conductance opening increases with WT and H101Q CLIC2 and is correlated with reduced FKBP/RyR association. The sub-conductance opening reduces RyR currents in the presence of WT CLIC2. In contrast, sub-conductance openings contribute to excess RyR “leak” with H101Q CLIC2. There are significant FKBP and RyR isoform-specific actions of CLIC2, rapamycin and FK506 on FKBP/RyR association. The results show that FKBPs do influence RyR gating and that this would contribute to excess Ca2+ release in one RyR channelopathy.
Publisher: Springer Science and Business Media LLC
Date: 1988
DOI: 10.1007/BF00581649
Abstract: Testing for correlations between different sets of genomic features is a fundamental task in genomics research. However, searching for overlaps between features with existing web-based methods is complicated by the massive datasets that are routinely produced with current sequencing technologies. Fast and flexible tools are therefore required to ask complex questions of these data in an efficient manner. This article introduces a new software suite for the comparison, manipulation and annotation of genomic features in Browser Extensible Data (BED) and General Feature Format (GFF) format. BEDTools also supports the comparison of sequence alignments in BAM format to both BED and GFF features. The tools are extremely efficient and allow the user to compare large datasets (e.g. next-generation sequencing data) with both public and custom genome annotation tracks. BEDTools can be combined with one another as well as with standard UNIX commands, thus facilitating routine genomics tasks as well as pipelines that can quickly answer intricate questions of large genomic datasets. BEDTools was written in C++. Source code and a comprehensive user manual are freely available at /bedtools aaronquinlan@gmail.com imh4y@virginia.edu Supplementary data are available at Bioinformatics online.
Publisher: Elsevier BV
Date: 04-1997
DOI: 10.1016/S0304-3940(97)00193-6
Abstract: In this report we demonstrate that the immunosuppressive drug, rapamycin, can reversibly activate the skeletal muscle ryanodine receptor calcium release channel (RyR) in terminal cisternae vesicles incorporated into planar lipid bilayers. This reveals a second mechanism of activation of RyRs by rapamycin. Irreversible channel activation and openings to subconductance levels are seen when rapamycin forms a complex with and removes the tightly bound 12 kDa FK506-binding protein (FKBP12) from the RyR. We show here that micromolar rapamycin activates RyRs which were previously 'stripped' of > 95% of their FKBP12s. Rapamycin caused a 6-fold increase in mean current, which was largely reversible, but no increase in the fraction of openings to subconductance levels. Therefore native RyRs, stripped of FKBP12, are directly activated by the macrocyclic lactone, rapamycin.
Publisher: Elsevier BV
Date: 02-2007
DOI: 10.1016/J.PHARMTHERA.2006.08.007
Abstract: This review addresses the potential use of the intracellular ryanodine receptor (RyR) Ca(2+) release channel as a therapeutic target in heart disease. Heart disease encompasses a wide range of conditions with the major contributors to mortality and morbidity being ischaemic heart disease and heart failure (HF). In addition there are many rare, but devastating conditions, some of which are either genetically linked to the RyR and its regulatory proteins or involve drug-induced modification of the proteins. The defects in Ca(2+) signalling vary with the nature of the heart disease and the stage in its progress and therefore specific corrections require different modifications of Ca(2+) signalling. Compounds that activate the RyR are potential inotropic agents to increase the Ca(2+) transient and strength of contraction. Compounds that reduce RyR activity are potentially useful in conditions where excess RyR activity initiates arrhythmias, or depletes the Ca(2+) store, as in end stage HF. It has recently been discovered that the cardio-protective action of the drug JTV519 can be attributed partly to its ability to stabilise the interaction between the RyR and the 12.6 kDa binding protein for the commonly used immunosuppressive drug FK506 (FKBP12.6, known as tacrolimus). This has established the credibility of the RyR as a therapeutic target. We explore the possibility that mutations causing the rare RyR-linked arrhythmias will open the door to identification of novel RyR-based therapeutic agents. The use of regulatory binding sites within the RyR complex or on its associated proteins as templates for drug design is discussed.
Publisher: Springer Science and Business Media LLC
Date: 15-05-1997
Abstract: The actions of D-myo-inositol 1,4,5-trisphosphate (IP3) and D-myo-inositol 1,3,4,5-tetrakisphospate (IP4) on small chloride (SCl) channels from rabbit skeletal muscle sarcoplasmic reticulum are reported. We find that the inositol polyphosphates (6-40 microM) are potent reversible blockers of SCl channels in lipid bilayers at -40 mV with >10(-5) m cis (cytoplasmic) Ca2+ when added to the cis, but not trans, chamber. IP3 or IP4 at 20 microM reduced the mean open time from 89 +/- 16 msec to 11 +/- 2 msec or to 8.0 +/- 1.0 msec respectively, by abolishing the longest time constant component in the open time distribution. Neither IP3 nor IP4 altered the six single-channel conductance levels. The fraction of low conductance events increased approximately 4-fold and the dwell time at the lower conductance levels increased approximately 3-fold. Channel gating was altered so that most transitions were between the closed level and an open level, in contrast to control channels which remained open for long periods with many transitions between the six open levels. The actions of the inositol polyphosphates were: (1) not prevented by 20 microg/ml cis heparin (an IP3 receptor blocker) (2) mimicked by 10 microM cis synthetic inositol polyphosphates, L-chiro-inositol 1,4,6-trisphosphate and L-chiro-inositol 1,4,6-trisphosphorothionate (which do not bind to IP3 receptors) (3) mimicked by cis additions of the polyanions heparin or hepran (20 microg/ml each) and vanadate (500 microM). The results suggest that an interaction between polyanions and SCl channels would allow the channels to be modulated in vivo by inositol polyphosphates.
Publisher: Elsevier BV
Date: 06-2012
DOI: 10.1016/J.BCP.2012.02.020
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 Ca(2+) release studies to determine that the binding site for GSTM2C is in ergent 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 Ca(2+) and improve contractility.
Publisher: Springer Science and Business Media LLC
Date: 22-02-2015
Publisher: Canadian Science Publishing
Date: 09-1987
DOI: 10.1139/Y87-288
Abstract: The effects of diazepam on internal membrane potential and action potentials and the isometric twitch and tetanus have been examined in rat fast-twitch (extensor digitorum longus) and slow-twitch (soleus) fibres. Low concentrations of the drug encountered during clinical usage (about 10 μM) had no effect on the membrane electrical properties or contractile properties of the fibres. Higher concentrations of diazepam (100–800 μM) induced changes in action potentials and excitation–contraction coupling but not in the resting membrane potential. After exposure to diazepam there was a rapid, concentration-dependent increase in twitch tension, which was attributed to an effect on excitation–contraction coupling, since the action potential and membrane potential were not altered. Soleus fibres were most sensitive to the potentiating action of diazepam. The decay of the tetanus was prolonged in both types of fibre, which indicated that diazepam blocked calcium uptake by the sarcoplasmic reticulum. Unexpectedly, the decay of isometric twitches was sensitive to diazepam only in soleus fibres, suggesting that calcium uptake was rate-limiting for tension relaxation in slow- but not fast-twitch fibres. The litude of the twitch and tetanus fell below control levels after prolonged exposure to diazepam, and there was a parallel reduction in action potential overshoot, especially during tetanic stimulation. Fast-twitch fibres were most susceptible to the depressant effect of diazepam.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 15-09-2017
Abstract: The chemotherapeutic anthracycline metabolite doxorubicinol (doxOL) has been shown to interact with and disrupt the function of the cardiac ryanodine receptor Ca
Publisher: Informa UK Limited
Date: 17-02-2011
DOI: 10.3109/03602532.2010.549134
Abstract: Glutathione transferases (GSTs) are generally recognized for their role in phase II detoxification reactions. However, it is becoming increasingly apparent that members of the GST family also have a erse range of other functions that are, in general, unrelated to detoxification. One such action is a specific inhibition of the cardiac isoform of the ryanodine receptor (RyR2) intracellular Ca(2+) release channel. In this review, we compare functional and physical interactions between members of the GST family, including GSTO1-1, GSTA1-1, and GSTM2-2, with RyR2 and with the skeletal isoform of the ryanodine receptor (RyR1). The active part of the muscle-specific GSTM2-2 is localized to its nonenzymatic C-terminal α-helical bundle, centered around α-helix 6. The GSTM2-2 binding site is in ergent region 3 (DR3 region) of RyR2. The sequence differences between the DR3 regions of RyR1 and RyR2 explain the specificity of the GSTs for one isoform of the protein. GSTM2-2 is one of the few known endogenous inhibitors of the cardiac RyR and is likely to be important in maintaining low RyR2 activity during diastole. We discuss interactions between a nonenzymatic member of the GST structural family, the CLIC-2 (type 2 chloride intracellular channel) protein, which inhibits both RyR1 and RyR2. The possibility that the GST and CLIC2 proteins bind to different sites on the RyR, and that different structures within the GST and CLIC proteins bind to RyR channels, is discussed. We conclude that the C-terminal part of GSTM2-2 may provide the basis of a therapeutic compound for use in cardiac disorders.
Publisher: Canadian Science Publishing
Date: 02-1987
DOI: 10.1139/Y87-048
Abstract: The action of the tranquilizer diazepam on rat skeletal muscle showed that relaxation of isometric twitches is controlled by different processes in extensor digitorum longus (fast-twitch) and soleus (slow-twitch) muscles. Diazepam caused an increase in the litude of twitches in fibres from both muscles but increased the twitch duration only in soleus. The litude of fused tetani were reduced in both muscles and the rate of relaxation after the tetanus slowed by as much as 34% when the litude of the tetanus was reduced by only 11%. The slower tetanic relaxation indicated that calcium uptake by the sarcoplasmic reticulum was slower than normal in slow- and fast-twitch fibres. We conclude therefore that calcium uptake by the sarcoplasmic reticulum is rate limiting for twitch relaxation in slow-twitch but not fast-twitch fibres and suggest that calcium binding to parvalbumin controls relaxation in the fast fibres.
Publisher: Wiley
Date: 05-1981
DOI: 10.1113/JPHYSIOL.1981.SP013693
Abstract: 1. Mouse extensor digitorum longus and soleus muscles respond to a sudden maintained increase in external K ion concentration with a fast contracture which inactivates and is followed by a slow contracture. 2. The slow contracture could not be selectively eliminated by altering the tonicity, ionic strength, anionic composition or buffer system of the external solution and depended only on the increase on external potassium concentration. The slow contracture differed from the fast K contracture in its time course, temperature sensitivity, fibre type dependence, and inactivation kinetics. The fast and slow contractures were similarly altered by changes in external anion species, by changes in external alent cations, and by the presence of 20 mM-caffeine. 3. The mechanism and functional significance of the slow contracture are obscure. The results suggest that its generation is not identical to that of the fast contracture, but may depend, in part, upon the normal activation processes.
Publisher: Rockefeller University Press
Date: 10-09-2007
Abstract: Ca2+ release from intracellular stores is controlled by complex interactions between multiple proteins. Triadin is a transmembrane glycoprotein of the junctional sarcoplasmic reticulum of striated muscle that interacts with both calsequestrin and the type 1 ryanodine receptor (RyR1) to communicate changes in luminal Ca2+ to the release machinery. However, the potential impact of the triadin association with RyR1 in skeletal muscle excitation–contraction coupling remains elusive. Here we show that triadin binding to RyR1 is critically important for rapid Ca2+ release during excitation–contraction coupling. To assess the functional impact of the triadin-RyR1 interaction, we expressed RyR1 mutants in which one or more of three negatively charged residues (D4878, D4907, and E4908) in the terminal RyR1 intraluminal loop were mutated to alanines in RyR1-null (dyspedic) myotubes. Coimmunoprecipitation revealed that triadin, but not junctin, binding to RyR1 was abolished in the triple (D4878A/D4907A/E4908A) mutant and one of the double (D4907A/E4908A) mutants, partially reduced in the D4878A/D4907A double mutant, but not affected by either in idual (D4878A, D4907A, E4908A) mutations or the D4878A/E4908A double mutation. Functional studies revealed that the rate of voltage- and ligand-gated SR Ca2+ release were reduced in proportion to the degree of interruption in triadin binding. Ryanodine binding, single channel recording, and calcium release experiments conducted on WT and triple mutant channels in the absence of triadin demonstrated that the luminal loop mutations do not directly alter RyR1 function. These findings demonstrate that junctin and triadin bind to different sites on RyR1 and that triadin plays an important role in ensuring rapid Ca2+ release during excitation–contraction coupling in skeletal muscle.
Publisher: Elsevier BV
Date: 2002
Publisher: Public Library of Science (PLoS)
Date: 09-09-2016
Publisher: Wiley
Date: 10-08-2006
DOI: 10.1111/J.1440-1681.2006.04441.X
Abstract: 1. Excitation-contraction coupling is broadly defined as the process linking the action potential to contraction in striated muscle or, more narrowly, as the process coupling surface membrane depolarization to Ca(2+) release from the sarcoplasmic reticulum. 2. We now know that excitation-contraction coupling depends on a macromolecular protein complex or 'calcium release unit'. The complex extends the extracellular space within the transverse tubule invaginations of the surface membrane, across the transverse tubule membrane into the cytoplasm and then across the sarcoplasmic reticulum membrane and into the lumen of the sarcoplasmic reticulum. 3. The central element of the macromolecular complex is the ryanodine receptor calcium release channel in the sarcoplasmic reticulum membrane. The ryanodine receptor has recruited a surface membrane L-type calcium channel as a 'voltage sensor' to detect the action potential and the calcium-binding protein calsequestrin to detect in the environment within the sarcoplasmic reticulum. Consequently, the calcium release channel is able to respond to surface depolarization in a manner that depends on the Ca(2+) load within the calcium store. 4. The molecular components of the 'calcium release unit' are the same in skeletal and cardiac muscle. However, the mechanism of excitation-contraction coupling is different. The signal from the voltage sensor to ryanodine receptor is chemical in the heart, depending on an influx of external Ca(2+) through the surface calcium channel. In contrast, conformational coupling links the voltage sensor and the ryanodine receptor in skeletal muscle. 5. Our current understanding of this amazingly efficient molecular signal transduction machine has evolved over the past 50 years. None of the proteins had been identified in the 1950s indeed, there was debate about whether the molecules involved were, in fact, protein. Nevertheless, a multitude of questions about the molecular interactions and structures of the proteins and their interaction sites remain to be answered and provide a challenge for the next 50 years.
Publisher: Wiley
Date: 16-10-2006
DOI: 10.1111/J.1440-1681.2006.04501.X
Abstract: 1. Excitation-contraction coupling in skeletal muscle is dependent on a physical interaction between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR). 2. A number of peptides derived from the II-III loop region of the DHPR have been shown to be functionally active in stimulating the release of calcium via RyR channels. Their function has been found to correlate with the presence of a basic helical region located at the N-terminus of the II-III loop. 3. The entire recombinant skeletal DHPR II-III loop is an efficient activator of RyR1 and RyR2. 4. The skeletal DHPR II-III loop is comprised of a series of a-helices, but its tertiary structure has been determined to be unstructured and flexible. 5. Fluorescence quenching experiments have been used to identify and measure the binding affinity of the II-III loop with fragments of the RyR.
Publisher: Springer Science and Business Media LLC
Date: 2000
Publisher: Wiley
Date: 08-1983
DOI: 10.1113/JPHYSIOL.1983.SP014802
Abstract: Asymmetrical charge movements (Q) were recorded from the voltage-cl ed ends of muscle fibres in extensor digitorum longus (e.d.l.) and soleus muscles from rats. Tetracaine (2 mM) was added to solutions to prevent contraction. In both muscles the relationship between Q and membrane potential (V) was S-shaped and could be described by the Boltzmann-type equation Q = Qm/(1 + exp[-(v - V)/k]) where Qm was the maximum charge, V the membrane potential at which Q = Qm/2, and k a 'slope factor'. On average, Qm was 5-6 times greater in e.d..l. than in soleus fibres and charge movement occurred at more negative potentials in soleus than in e.d.l. fibres, V being -36.7 mV in the former and -19.0 mV in the latter, a difference of about 18 mV. The threshold for contraction, determined using a two-electrode voltage cl , was more negative in soleus than in e.d.l. fibres. For 500 ms depolarizations, the difference was 12 mV. The relationship between tension and membrane potential during potassium contractures was S-shaped and, when fitted by the Boltzmann-type equation, gave V values of -25 mV for soleus and -14 mV for e.d.l. fibres. In paraplegic rats, the threshold for contraction in soleus fibres shifted about 12 mV to more positive potentials, but there was no change in e.d.l. fibres so that there was no significant difference between the two muscles. In paraplegic rats the relationship between tension and membrane potential during potassium contractures also shifted to more positive potentials in soleus fibres, whereas there was no change in e.d.l. fibres. These changes in the voltage sensitivity of contractile activation in soleus fibres from paraplegic rats were associated with a parallel shift in the voltage sensitivity of charge movement so that the average V shifted from -36.7 mV in normal rats to a value of -14.2 mV in paraplegic rats. There was also a four-fold increase in Qm in soleus fibres from paraplegic rats. The difference between the voltage sensitivity of contractile activation and charge movement in e.d.l. and soleus fibres in normal rats supports the hypothesis that the two are closely related: even stronger support comes from the observation of the parallel shift in the voltage sensitivity of contractile activation and charge movement in soleus fibres in paraplegic rats.
Publisher: Elsevier BV
Date: 03-2008
Publisher: Portland Press Ltd.
Date: 11-12-2007
DOI: 10.1042/BJ20060902
Abstract: To explain the mechanism of pathogenesis of channel disorder in MH (malignant hyperthermia), we have proposed a model in which tight interactions between the N-terminal and central domains of RyR1 (ryanodine receptor 1) stabilize the closed state of the channel, but mutation in these domains weakens the interdomain interaction and destabilizes the channel. DP4 (domain peptide 4), a peptide corresponding to residues Leu2442–Pro2477 of the central domain, also weakens the domain interaction and produces MH-like channel destabilization, whereas an MH mutation (R2458C) in DP4 abolishes these effects. Thus DP4 and its mutants serve as excellent tools for structure–function studies. Other MH mutations have been reported in the literature involving three other amino acid residues in the DP4 region (Arg2452, Ile2453 and Arg2454). In the present paper we investigated the activity of several mutants of DP4 at these three residues. The ability to activate ryanodine binding or to effect Ca2+ release was severely diminished for each of the MH mutants. Other substitutions were less effective. Structural studies, using NMR analysis, revealed that the peptide has two α-helical regions. It is apparent that the MH mutations are clustered at the C-terminal end of the first helix. The data in the present paper indicates that mutation of residues in this region disrupts the interdomain interactions that stabilize the closed state of the channel.
Publisher: The Company of Biologists
Date: 2014
DOI: 10.1242/JCS.156760
Abstract: We report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca2+]. Basal leak from the sarcoplasmic reticulum (SR) is required for normal Ca2+ handling, but excess diastolic Ca2+ leak attributed to oxidative stress is thought to lower RyR2 threshold for spontaneous SR Ca2+ release to induce arrhythmia in pathological situations. Therefore we examined RyR2 response to luminal [Ca2+] under reducing or oxidising cytoplasmic redox conditions. Unexpectedly as luminal [Ca2+] increased from 0.1–1.5 mM RyR2 activity declined when pretreated with cytoplasmic 1 mM DTT, or GSH∶GSSG buffered to a “healthy” reduced cytoplasmic redox potential (−220 mV). Conversely, with 20 µM cytoplasmic 4,4′-DTDP, or redox buffered to an oxidising −180 mV, RyR2 activity increased with increasing luminal [Ca2+]. The luminal redox potential was constant at −180 mV in each case. These responses to luminal Ca2+ were maintained with 2 mM Na2ATP or 5 mM MgATP (1 mM free Mg2+). Overall the results suggest that the redox potential in the RyR2 junctional microdomain is normally more oxidised than the bulk cytoplasm.
Publisher: Springer Science and Business Media LLC
Date: 06-1982
DOI: 10.1007/BF00711941
Abstract: This paper theoretically simulated (using DFT and TD-DFT in N,N-dimethylformamide (DMF) solvent) the photodynamic properties of three non-metallic dye molecules with D-π-A₁-π-A₂ structure. The total photoelectric conversion efficiency (PCE) could be evaluated by the following parameters: the geometric structures, the electronic structures, and the absorption spectra, the analyses of charge difference density (CDD) and natural bond orbitals (NBO), the analyses of ionization potential (IP) and electron affinity (EA) from electronic contribution capacity, the reorganization energies (λh, λe, and λtotal), and the chemical reaction parameter (h,
Publisher: Springer Science and Business Media LLC
Date: 10-06-2009
DOI: 10.1007/S00249-009-0494-1
Abstract: Homer proteins have recently been identified as novel high-affinity ligands that modulate ryanodine receptor (RyR) Ca(2+) release channels in heart and skeletal muscle, through an EVH1 domain which binds to proline-rich regions in target proteins. Many Homer proteins can also self-associate through a coiled-coil domain that allows their multimerisation. In other tissues, especially neurons, Homer anchors proteins embedded in the surface membrane to the Ca(2+) release channel in the endoplasmic reticulum and can anchor membrane or cytosolic proteins to the cytoskeleton. Although this anchoring aspect of Homer function has not been extensively investigated in muscle, there are consensus sequences for Homer binding in the RyR and on many of the proteins that it interacts with in the massive RyR ion channel complex. In this review we explore the potential of Homer to contribute to a variety of cell processes in muscle and neurons that also involve RyR channels.
Publisher: Springer Science and Business Media LLC
Date: 12-1982
DOI: 10.1007/BF01868650
Abstract: Hairy cell leukemia (HCL) in Western patients typically expresses CD19, CD20, CD11c, CD25, HLA-DR, and IgG/lambda and lacks expression of CD5 and CD10. The immunophenotype is in contrast to Japanese HCL which typically expresses CD5 and CD10. Western and Japanese HCL also differ in their clinical presentation and response to treatment with alpha-interferon. We report a case of non-Japanese HCL which presented typically with pancytopenia however, the immunophenotype was atypical with expression of CD10 and CDw75. CDw75 expression has not previously been described in either Japanese or non-Japanese HCL. The patient achieved a marked partial pathologic response and complete clinical response to treatment with cladaribine phosphate.
Publisher: Wiley
Date: 29-09-2003
DOI: 10.1046/J.1440-1681.2003.03904.X
Abstract: 1. The ryanodine receptor (RyR) is the Ca2+ release channel in the sarcoplamic reticulum of skeletal and cardiac muscle and is essential for respiration and heart beat. The RyR channel releases Ca2+ from intracellular stores in a variety of other cell types, where it normally coexists with the inositiol 1,4,5-trisphosphate receptor (IP3R). The RyR and IP3R, forming a superfamily of homotetrameric ligand-gated intracellular Ca2+ channels, serve discrete functions: they can be located in independent Ca2+ stores with different activation mechanisms and can be coupled to different signalling pathways. 2. Although functional characteristics of the RyR have been investigated intensely, there remain major gaps in our knowledge about the structure of the protein, its ion-conducting pore, its ligand-binding sites and sites supporting the many protein rotein interactions that underlie the in vivo function of the channel. 3. Of particular importance are the transmembrane segments that form the membrane-spanning domain of the protein and the pore, define the conductance and selectivity of the channel and dictate the cytoplasmic and luminal domains and the overall protein structure. Hydropathy profiles predict between four and 12 transmembrane segments. One popular model shows four transmembrane segments in the C-terminal one-tenth of the protein. However, there is substantial evidence for a larger number of membrane-spanning segments located in both the C-terminal and central parts of the protein. 4. A model of the RyR pore based on the Streptomyces lividans KcsA channel structure is presented. Protein rotein interactions between the RyR and other regulatory proteins, as well as within the RyR subunit, are discussed.
Publisher: Wiley
Date: 07-09-2012
DOI: 10.1096/FJ.12-211334
Abstract: Excitation-contraction (EC) coupling in skeletal muscle depends on protein interactions between the transverse tubule dihydropyridine receptor (DHPR) voltage sensor and intracellular ryanodine receptor (RyR1) calcium release channel. We present novel data showing that the C-terminal 35 residues of the β(1a) subunit adopt a nascent α-helix in which 3 hydrophobic residues align to form a hydrophobic surface that binds to RyR1 isolated from rabbit skeletal muscle. Mutation of the hydrophobic residues (L496, L500, W503) in peptide β(1a)V490-M524, corresponding to the C-terminal 35 residues of β(1a), reduced peptide binding to RyR1 to 15.2 ± 7.1% and prevented the 2.9 ± 0.2-fold activation of RyR1 by 10 nM wild-type peptide. An upstream hydrophobic heptad repeat implicated in β(1a) binding to RyR1 does not contribute to RyR1 activation. Wild-type β(1a)A474-A508 peptide (10 nM), containing heptad repeat and hydrophobic surface residues, increased RyR1 activity by 2.3 ± 0.2- and 2.2 ± 0.3-fold after mutation of the heptad repeat residues. We conclude that specific hydrophobic surface residues in the 35 residue β(1a) C-terminus bind to RyR1 and increase channel activity in lipid bilayers and thus may support skeletal EC coupling.
Publisher: Bentham Science Publishers Ltd.
Date: 05-2011
DOI: 10.2174/138945011795378595
Abstract: The ryanodine receptor (RyR) calcium release channel is an essential intracellular ion channel that is central to Ca(2+) signaling and contraction in the heart and skeletal muscle. The rapid release of Ca(2+) from the internal sarcoplasmic reticulum Ca(2+) stores through the RyR during excitation-contraction coupling is facilitated by the unique arrangement of the surface and sarcoplasmic reticulum membrane systems. Debilitating and sometimes fatal skeletal and cardiomyopathies result from changes in RyR activity that disrupt normal Ca(2+) signaling. Such changes can be caused by point mutations in many different regions of the RyR protein or acquired as a result of stress associated with exercise, heart failure, age or drugs. In general, both inherited and acquired changes include an increase in RyR channel activity. Because of its central function, the RyR is a potential therapeutic target for the inherited disorders and many of the acquired disorders. The RyR is currently used as a therapeutic target in malignant hyperthermia where dantrolene is effective and to relieve ventricular arrhythmia, with the use of JTV519 and flecainide. These drugs show that the RyR is a valid therapeutic target, but have side effects that prevent their chronic use. Thus there is an urgent need for the development of skeletal and cardiac specific drugs to treat these erse muscle disorders. In this review, we discuss the mutations that cause skeletal myopathies and cardiac arrhythmias and how these mutations pinpoint residues within the RyR protein that are functionally significant and might be developed as targets for therapeutic drugs.
Publisher: Elsevier BV
Date: 12-2019
Publisher: Wiley
Date: 09-2001
DOI: 10.1111/J.1469-7793.2001.T01-1-00715.X
Abstract: 1. Phosphate ions (P(i)) enter intracellular Ca2+ stores and precipitate Ca2+. Since transport pathways for P(i) across the membrane of intracellular calcium stores have not been identified and anion channels could provide such a pathway, we have examined the P(i) conductance of single anion channels from the sarcoplasmic reticulum (SR) of rabbit skeletal muscle using the lipid bilayer technique. 2. Two anion channels in skeletal muscle SR, the small conductance (SCl) and big conductance (BCl) chloride channels, were both found to have a P(i) conductance of 10 pS in 50 mM P(i). The SCl channel is a alent anion channel which can pass HPO4(2-) as well as SO4(2-) (60 pS in 100 mM free SO4(2-)). The BCl channel is primarily a monovalent anion channel. The SCl and BCl channels are permeable to a number of small monovalent anions, showing minor selectivity between Cl-, I- and Br- (Cl- > I- > Br-) and relative impermeability to cations and large polyatomic anions (Cs+, Na+, choline+, Tris+, Hepes- and CH3O3S-). 3. The P(i) conductance of SCl and BCl channels suggests that both channel types could sustain the observed P(i) fluxes across the SR membrane. Comparison of the blocking effects of the phosphonocarboxylic acids, ATP and DIDS, on the anion channels with their effects on P(i) transport suggests that the SCl channel is the more likely candidate for the SR P(i) transport mechanism. 4. The SCl channel, with previously unknown function, provides a regulated pathway for P(i) across the SR membrane which would promote P(i) entry and thereby changes in the rapidly releasable Ca2+ store during onset and recovery from muscle fatigue. Anion channels may provide a pathway for P(i) movement into and out of Ca2+ stores in general.
Publisher: Portland Press Ltd.
Date: 09-08-2005
DOI: 10.1042/BJ20042113
Abstract: The recently discovered CLIC-2 protein (where CLIC stands for chloride intracellular channel), which belongs to the ubiquitous glutathione transferase structural family and is expressed in the myocardium, is a regulator of native cardiac RyR2 (ryanodine receptor 2) channels. Here we show that recombinant CLIC-2 increases [3H]ryanodine binding to native and purified RyR channels, enhances substate activity in in idual channels, increases the number of rare coupled gating events between associated RyRs, and reduces activation of the channels by their primary endogenous cytoplasmic ligands, ATP and Ca2+. CLIC-2 (0.2–10 μM) added to the cytoplasmic side of RyR2 channels in lipid bilayers depressed activity in a reversible, voltage-independent, manner in the presence of activating (10–100 μM) or sub-activating (100 nM) cytoplasmic Ca2+ concentrations. Although the number of channel openings to all levels was reduced, the fraction and duration of openings to substate levels were increased after exposure to CLIC-2. CLIC-2 reduced increases in activity induced by ATP or adenosine 5′-[β,γ-imido]triphosphate. Depression of channel activity by CLIC-2 was greater in the presence of 100 μM cytoplasmic Ca2+ than with 100 nM or 10 μM Ca2+. Further, CLIC-2 prevented the usual ∼50-fold increase in activity when the cytoplasmic Ca2+ concentration was increased from 100 nM to 100 μM. The results show that CLIC-2 interacts with the RyR protein by a mechanism that does not require oxidation, but is influenced by a conserved Cys residue at position 30. CLIC-2 is one of only a few cytosolic inhibitors of cardiac RyR2 channels, and may suppress their activity during diastole and during stress. CLIC-2 provides a unique probe for substate activity, coupled gating and ligand-induced activation of cardiac RyR channels.
Publisher: Elsevier BV
Date: 06-2001
Publisher: The Royal Society
Date: 05-2023
Abstract: Cardiac ryanodine receptors (RyR2) release the Ca 2+ from intracellular stores that is essential for cardiac myocyte contraction. The ion channel opening is tightly regulated by intracellular factors, including the FK506 binding proteins, FKBP12 and FKBP12.6. The impact of these proteins on RyR2 activity and cardiac contraction is debated, with often apparently contradictory experimental results, particularly for FKBP12. The isoform that regulates RyR2 has generally been considered to be FKBP12.6, despite the fact that FKBP12 is the major isoform associated with RyR2 in some species and is bound in similar proportions to FKBP12.6 in others, including sheep and humans. Here, we show time- and concentration-dependent effects of adding FKBP12 to RyR2 channels that were partly depleted of FKBP12/12.6 during isolation. The added FKBP12 displaced most remaining endogenous FKBP12/12.6. The results suggest that FKBP12 activates RyR2 with high affinity and inhibits RyR2 with lower affinity, consistent with a model of negative cooperativity in FKBP12 binding to each of the four subunits in the RyR tetramer. The easy dissociation of some FKBP12/12.6 could dynamically alter RyR2 activity in response to changes in in vivo regulatory factors, indicating a significant role for FKBP12/12.6 in Ca 2+ signalling and cardiac function in healthy and diseased hearts. This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’.
Publisher: The Royal Society
Date: 05-2023
Abstract: Skeletal and cardiac muscle excitation–contraction coupling commences with Na v 1.4/Na v 1.5-mediated, surface and transverse (T-) tubular, action potential generation. This initiates feedforward , allosteric or Ca 2+ -mediated, T-sarcoplasmic reticular (SR) junctional, voltage sensor-Cav1.1/Cav1.2 and ryanodine receptor-RyR1/RyR2 interaction. We review recent structural, physiological and translational studies on possible feedback actions of the resulting SR Ca 2+ release on Na v 1.4/Na v 1.5 function in native muscle. Finite-element modelling predicted potentially regulatory T-SR junctional [Ca 2+ ] TSR domains. Na v 1.4/Na v 1.5, III-IV linker and C-terminal domain structures included Ca 2+ and/or calmodulin-binding sites whose mutations corresponded to specific clinical conditions. Loose-patch-cl ed native murine skeletal muscle fibres and cardiomyocytes showed reduced Na + currents ( I Na ) following SR Ca 2+ release induced by the Epac and direct RyR1/RyR2 activators, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate and caffeine, abrogated by the RyR inhibitor dantrolene. Conversely, dantrolene and the Ca 2+ -ATPase inhibitor cyclopiazonic acid increased I Na . Experimental, catecholaminergic polymorphic ventricular tachycardic RyR2-P2328S and metabolically deficient Pgc1β −/− cardiomyocytes also showed reduced I Na accompanying [Ca 2+ ] i abnormalities rescued by dantrolene- and flecainide-mediated RyR block. Finally, hydroxychloroquine challenge implicated action potential (AP) prolongation in slowing AP conduction through modifying Ca 2+ transients. The corresponding tissue/organ preparations each showed pro-arrhythmic, slowed AP upstrokes and conduction velocities. We finally extend discussion of possible Ca 2+ -mediated effects to further, Ca 2+ , K + and Cl − , channel types. This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’.
Publisher: Mary Ann Liebert Inc
Date: 10-2008
Abstract: The type 2 chloride intracellular channel, CLIC-2, is a member of the glutathione S-transferase structural family and a suppressor of cardiac ryanodine receptor (RyR2) Ca2+ channels located in the membrane of the sarcoplasmic reticulum (SR). Modulators of RyR2 activity can alter cardiac contraction. Since both CLIC-2 and RyR2 are modified by redox reactions, we speculated that the action of CLIC-2 on RyR2 may depend on redox potential. We used a GSH:GSSG buffer system to produce mild changes in redox potential to influence redox sensors in RyR2 and CLIC-2. RyR2 activity was modified only when both luminal and cytoplasmic solutions contained the GSH:GSSG buffer and the effects were reversed by removing the buffer from one of the solutions. Channel activity increased with an oxidizing redox potential and decreased when the potential was more reducing. Addition of cytoplasmic CLIC-2 inhibited RyR2 with oxidizing redox potentials, but activated RyR2 under reducing conditions. The results suggested that both RyR2 and CLIC-2 contain redox sensors. Since cardiac ischemia involves a destructive Ca2+ overload that is partly due to oxidation-induced increase in RyR2 activity, we speculate that the properties of CLIC-2 place it in an ideal position to limit ischemia-induced cellular damage in cardiac muscle.
Publisher: Wiley
Date: 28-08-2018
Abstract: Ryanodine receptor (RyR) Ca
Publisher: Wiley
Date: 09-1979
Abstract: A 320-bp fragment of the Arabidopsis cab2 promoter is sufficient to mediate transcriptional regulation by both phytochrome and the circadian clock. We fused this promoter fragment to the firefly luciferase (Luc) gene to create a real-time reporter for regulated gene expression in intact plants. Cab2::Luc transcript accumulated in the expected patterns and luciferase activity was closely correlated to cab2::Luc mRNA abundance in both etiolated and green seedlings. The concentration of the bulk of luciferase protein did not reflect these patterns but maintained a relatively constant level, implying that a post-translational mechanism(s) leads to the high- litude regulation of luciferase activity. We used a low-light video imaging system to establish that luciferase bioluminescence in vivo accurately reports the temporal and spatial regulation of cab2 transcription in single seedlings. The unique qualities of the firefly luciferase system allowed us to monitor regulated gene expression in real time in in idual multicellular organisms. This noninvasive marker for temporal regulation at the molecular level constitutes a circadian phenotype, which may be used to isolate mutants in the circadian clock.
Publisher: Elsevier BV
Date: 04-2009
DOI: 10.1016/J.BCP.2008.12.024
Abstract: The muscle specific glutathione transferase GSTM2-2 inhibits the activity of cardiac ryanodine receptor (RyR2) calcium release channels with high affinity and activates skeletal RyR (RyR1) channels with lower affinity. To determine which overall region of the GSTM2-2 molecule supports binding to RyR2, we examined the effects of truncating GSTM2-2 on its ability to alter Ca(2+) release from sarcoplasmic reticulum (SR) vesicles and RyR channel activity. The C-terminal half of GSTM2-2 which lacks the critical GSH binding site supported the inhibition of RyR2, but did not support activation of RyR1. Smaller fragments of GSTM2-2 indicated that the C-terminal helix 6 was crucial for the action of GSTM2-2 on RyR2. Only fragments containing the helix 6 sequence inhibited Ca(2+) release from cardiac SR. Single RyR2 channels were strongly inhibited by constructs containing the helix 6 sequence in combination with adjacent helices (helices 5-8 or 4-6). Fragments containing helices 5-6 or helix 6 sequences alone had less well-defined effects. Chemical cross-linking indicated that C-terminal helices 5-8 bound to RyR2, but not RyR1. Structural analysis with circular dichroism showed that the helical content was greater in the longer helix 6 containing constructs, while the helix 6 sequence alone had minimal helical structure. Therefore the active centre of GSTM2-2 for inhibition of cardiac RyR2 involves the helix 6 sequence and the helical nature of this region is essential for its efficacy. GSTM2-2 helices 5-8 may provide the basis for RyR2-specific compounds for experimental and therapeutic use.
Publisher: Elsevier BV
Date: 05-2009
DOI: 10.1016/J.CECA.2009.03.006
Abstract: Calcium signaling in myocytes is dependent on the cardiac ryanodine receptor (RyR2) calcium release channel and the calcium buffering protein in the sarcoplasmic reticulum, cardiac calsequestrin (CSQ2). The overall properties of CSQ2 and its regulation of RyR2 have not been explored in detail or directly compared with skeletal CSQ1 and its regulation of the skeletal RyR1, with physiological ionic strength and Ca(2+) concentrations. We find that there are major differences between the two isoforms under these physiological conditions. Ca(2+) binding to CSQ2 is 50% lower than to CSQ1. Only approximately 30% of CSQ2 is bound to cardiac junctional face membrane (JFM), compared with approximately 70% of CSQ1 and the ratio of CSQ2 to RyR2 is only 50% of the CSQ1/RyR1 ratio. Chemical crosslinking shows that CSQ2 is mostly monomer/dimer, while CSQ1 is mostly polymerized. In single channel lipid bilayer experiments, CSQ2 monomers and/or dimers increase the open probability of both RyR1 and RyR2 channels, while CSQ1 polymers decrease the activity of RyR1. We speculate that CSQ2 facilitates high rates of Ca(2+) release through RyR2 during systole, while CSQ1 curtails RyR1 opening in response to a single action potential to maintain Ca(2+) and allow repeated Ca(2+) release and graded activation with increased stimulation frequency.
Publisher: Elsevier BV
Date: 2008
DOI: 10.1016/J.BIOCEL.2007.12.019
Abstract: We show that a glutathione transferase (GST) protein, which is recognised by an antibody against the muscle-specific human GSTM2-2 (hGSTM2-2), is associated with the lumen of the sarcoplasmic reticulum (SR) of cardiac muscle, but not skeletal muscle. We further show that hGSTM2-2 modifies both cardiac and skeletal ryanodine receptor (RyR) activity when it binds to the luminal domain of the RyR channel complex. The properties of hGSTM2-2 were compared with those of the calsequestrin (CSQ), a Ca(2+) binding protein also present in the lumen of the SR which, like GSTM2-2, contains a thioredoxin-fold structure and modifies RyR activity (Wei, L., Varsanyi, M., Dulhunty, A. F., Beard, N. A. (2006). The Biophysical Journal, 91, 1288-1301). The glutathione transferase activity of hGSTM2-2 is strong, while CSQ is essentially inactive. Conversely CSQ is a strong Ca(2+) binder, but hGSTM2-2 is not. The effects of luminal hGSTM2-2 on RyR activity differ from those of CSQ in that hGSTM2-2 activates RyRs by increasing their open probability and conductance and the effects are independent of luminal Ca(2+) concentration. The results suggest that GSTM2-2 can interact with specific luminal sites on the RyR complex and that the interaction is likely to be within the pore of the RyR channel. The differences between the effects of CSQ and hGSTM2-2 suggest that the thioredoxin fold is not a major determinant of the luminal actions of either protein. The results indicate that GSTM2-2 is a novel luminal regulator of the RyR channels in the heart.
Publisher: Elsevier BV
Date: 04-1988
Publisher: Springer Science and Business Media LLC
Date: 05-1998
Abstract: The reactive disulfide 4,4'-dithiodipyridine (4,4' DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca2+] of 10(-7) M (in the absence of Mg2+ and ATP), increased within approximately 1 min of addition of 1 mM 4,4'-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mM cis Mg2+ (10(-7) M cis Ca2+) or by 10 mM cis Mg2+ (10(-3) M cis Ca2+), or activated by 4 mM ATP (10(-7) M cis Ca2+), also responded to 1 mM cis 4,4'-DTDP with activation and then loss of activity. Po and mean open time (T(o)) of the maximally activated channels were lower in the presence of Mg2+ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mM 4,4'-DTDP in channels inhibited by Mg2+, and the previously reported enhanced activation by 4,4'-DTDP in channels activated by Ca2+ or caffeine (Eager et al., 1997), the activation produced by 1 mM cis 4,4'-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation.
Publisher: Springer Science and Business Media LLC
Date: 02-1999
Abstract: The location of reactive cysteine residues on the ryanodine receptor (RyR) calcium release channel was assessed from the changes in channel activity when oxidizing or reducing reagents were added to the luminal or cytoplasmic solution. Single sheep cardiac RyRs were incorporated into lipid bilayers with 10(-7) m cytoplasmic Ca2+. The thiol specific-lipophilic-4,4'-dithiodipyridine (4,4'-DTDP, 1 mm), as well as the hydrophilic thimerosal (1 mm), activated and then inhibited RyRs from either the cis (cytoplasmic) or trans (luminal) solutions. Activation was associated with an increase in the (a) mean channel open time and (b) number of exponential components in the open time distribution from one ( approximately 2 msec) to three (approximately 1 msec approximately 7 msec approximately 15 msec) in channels activated by trans 4,4'-DTDP or cis or trans thimerosal. A longer component (approximately 75 msec) appeared with cis 4, 4'-DTDP. Activation by either oxidant was reversed by the thiol reducing agent, dithiothreitol. The results suggest that three classes of cysteines are available to 4,4'-DTDP or thimerosal, SHa or SHa* activating the channel and SHi closing the channel. SHa is either distributed over luminal and cytoplasmic RyR domains, or is located within the channel pore. SHi is also located within the transmembrane domain. SHa* is located on the cytoplasmic domain of the protein.
Publisher: Elsevier BV
Date: 06-2011
Publisher: Springer Science and Business Media LLC
Date: 20-05-2009
DOI: 10.1007/S00249-009-0467-4
Abstract: Voltage-gated Ca(2+) channels (VGCCs) are involved in a number of excitatory processes in the cell that regulate muscle contraction, neurotransmitter release, gene regulation, and neuronal migration. They consist of a central pore-forming alpha(1) subunit together with a number of associated auxiliary subunits including a cytoplasmic beta subunit. With the aid of X-ray crystallography, it has been found that the beta subunits of VGCCs (beta(2a), beta(3), and beta(4)) interact strongly with the I-II loop of the pore-forming alpha(1) subunit. Here we discuss the potential interaction sites of beta(1a) with its alpha(1) subunit as well as the skeletal ryanodine receptor. We suggest that not only can beta(1a) interact with the alpha(1) subunit I-II loop, but more subtle interactions may be possible through the II-III loop via the beta(1a) SH3 domain. Such findings could have important implications with respect to EC coupling.
Publisher: Elsevier BV
Date: 08-2004
Publisher: Wiley
Date: 04-1977
DOI: 10.1113/JPHYSIOL.1977.SP011788
Abstract: 1. The passive electrical properties of frog skeletal muscle fibres have been measured at a number of different sarcomere lengths (from 2-1 to 4-0 micron). The geometrical outline of each fibre was determined from optical cross-sections and sarcomere length was measured by laser beam diffraction. 2. When fibres were stretched to long sarcomere lengths the membrane capacity, Cm, of both normal and detubulated (glycerol-treated) fibres was significantly less than the Cm of fibres at rest length. A significant reduction in membrane conductance of fibres held at long sarcomere lengths was only seen with detubulated fibres. 3. Membrane capacity and membrane conductance have a significant dependence on the cross-sectional area of normal fibres but are independent of cross-sectional area after detubulation. 4. It has been shown that membrane geometry depends on the sarcomere length of the fibre and it is suggested that the passive membrane properties are related to sarcomere length because they depend on membrane geometry. 5. The specific membrane capacity, calculated from the data from detubulated fibres, is 0-8 micronF/cm2. 6. The internal resistivity, Ri, of normal fibres, also depends on sarcomere length between 2-1 and 3-0 micron. At a sarcomere length of 2-1 micron the average Ri is 122 +/- 3 omega. cm (mean +/- S.E. of mean) and at a sarcomere length of 3-0 micron the average Ri is 210 +/- 17 omega. cm (mean +/- S.E. of mean). No further increase in Ri was observed with further increases in sarcomere length.
Publisher: Springer Science and Business Media LLC
Date: 10-1980
DOI: 10.1007/BF01869590
Publisher: Public Library of Science (PLoS)
Date: 24-08-2012
Publisher: Wiley
Date: 25-02-2009
DOI: 10.1111/J.1440-1681.2008.05130.X
Abstract: 1. The dihydropyridine receptor (DHPR) II-III loop is an intrinsically unstructured region made up of alpha-helical and beta-turn secondary structure elements with the N and C termini in close spatial proximity. 2. The DHPR II-III loop interacts in vitro with a ryanodine receptor (RyR) 1 SPRY domain through alpha-helical segments located in the A and B regions. Mutations within the A and B regions in the DHPR II-III loop alter the binding affinity to the SPRY2 domain. 3. The A and C peptides derived from DHPR II-III loop show negative cooperativity in binding to the SPRY2 domain. 4. The SPRY2 domain of the RyR1 (1085-1208) forms a beta-sheet sandwich structure flanked by variable loop regions. An acidic loop region of SPRY2 (1107-1121) forms part of a negatively charged cleft that is implicated in the binding of the DHPR II-III loop. 5. The mutant E1108A located in the negatively charged loop of SPRY2 reduces the binding affinity to the DHPR II-III loop.
Publisher: Wiley
Date: 23-04-2012
DOI: 10.1111/J.1440-1681.2012.05704.X
Abstract: The contractile function of the heart requires the release of Ca(2+) from intracellular Ca(2+) stores in the sarcoplasmic reticulum (SR) of cardiac muscle cells. The efficacy of Ca(2+) release depends on the amount of Ca(2+) loaded into the Ca(2+) store and the way in which this 'Ca(2+) load' influences the activity of the cardiac ryanodine receptor Ca(2+) release channel (RyR2). The effects of the Ca(2+) load on Ca(2+) release through RyR2 are facilitated by: (i) the sensitivity of RyR2 itself to luminal Ca(2+) concentrations and (ii) interactions between the cardiac Ca(2+) -binding protein calsequestrin (CSQ) 2 and RyR2, transmitted through the 'anchoring' proteins junctin and/or triadin. Mutations in RyR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT) and sudden cardiac death. The tachycardia is associated with changes in the sensitivity of RyR2 to luminal Ca(2+) . Triadin-, junctin- or CSQ-null animals survive, but their longevity and ability to tolerate stress is compromised. These studies reveal the importance of the proteins in normal muscle function, but do not reveal the molecular nature of their functional interactions, which must be defined before changes in the proteins leading to CPVT and heart disease can be understood. Herein, we discuss known interactions between the RyR, triadin, junctin and CSQ with emphasis on the cardiac isoforms of the proteins. Where there is little known about the cardiac isoforms, we discuss evidence from skeletal isoforms.
Publisher: Springer Science and Business Media LLC
Date: 06-2000
Publisher: Portland Press Ltd.
Date: 24-01-2005
DOI: 10.1042/BJ20041152
Abstract: A physical association between the II–III loop of the DHPR (dihydropryidine receptor) and the RyR (ryanodine receptor) is essential for excitation–contraction coupling in skeletal, but not cardiac, muscle. However, peptides corresponding to a part of the II–III loop interact with the cardiac RyR2 suggesting the possibility of a physical coupling between the proteins. Whether the full II–III loop and its functionally important ‘C’ region (cardiac DHPR residues 855–891 or skeletal 724–760) interact with cardiac RyR2 is not known and is examined in the present study. Both the cardiac DHPR II–III loop (CDCL) and cardiac peptide (Cc) activated RyR2 channels at concentrations & nM. The skeletal DHPR II–III loop (SDCL) activated channels at ≤100 nM and weakly inhibited at ≥1 μM. In contrast, skeletal peptide (Cs) inhibited channels at all concentrations when added alone, or was ineffective if added in the presence of Cc. Ca2+-induced Ca2+ release from cardiac sarcoplasmic reticulum was enhanced by CDCL, SDCL and the C peptides. The results indicate that the interaction between the II–III loop and RyR2 depends critically on the ‘A’ region (skeletal DHPR residues 671–690 or cardiac 793–812) and also involves the C region. Structure analysis indicated that (i) both Cs and Cc are random coil at room temperature, but, at 5 °C, have partial helical regions in their N-terminal and central parts, and (ii) secondary-structure profiles for CDCL and SDCL are similar. The data provide novel evidence that the DHPR II–III loop and its C region interact with cardiac RyR2, and that the ability to interact is not isoform-specific.
Publisher: Elsevier BV
Date: 1992
Start Date: 2017
End Date: 2020
Funder: National Health and Medical Research Council
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End Date: 2016
Funder: National Health and Medical Research Council
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