A bivalent remipede toxin promotes calcium release via ryanodine receptor activation.

Multivalent ligands of ion channels have proven to be both very rare and highly valuable in yielding unique insights into channel structure and pharmacology. Here, we describe a bivalent peptide from the venom of Xibalbanus tulumensis, a troglobitic arthropod from the enigmatic class Remipedia, that causes persistent calcium release by activation of ion channels involved in muscle contraction. The high-resolution solution structure of φ-Xibalbin3-Xt3a reveals a tandem repeat arrangement of inhibitor-cysteine knot (ICK) domains previously only found in spider venoms. The individual repeats of Xt3a share sequence similarity with a family of scorpion toxins that target ryanodine receptors (RyR). Single-channel electrophysiology and quantification of released Ca2+ stores within skinned muscle fibers confirm Xt3a as a bivalent RyR modulator. Our results reveal convergent evolution of RyR targeting toxins in remipede and scorpion venoms, while the tandem-ICK repeat architecture is an evolutionary innovation that is convergent with toxins from spider venoms.

Secretoneurin Is an Endogenous Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor That Attenuates Ca2+-Dependent Arrhythmia.

BACKGROUND: Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, we hypothesized that upregulation of SN in patients protects against cardiomyocyte mechanisms of arrhythmia. METHODS: Circulating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia-induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9-induced overexpression. Interactions between SN and CaMKIIδ were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on Ca2+ homeostasis examined by fluorescence (fluo-4) and patch-clamp recordings in isolated cardiomyocytes. RESULTS: SN levels were elevated in patients with CPVT and following ventricular arrhythmia-induced cardiac arrest. In contrast to NT-proBNP (N-terminal pro-B-type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9-induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKIIδ. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKIIδ in Langendorff hearts and inhibited CaMKIIδ-dependent RyR phosphorylation. In line with CaMKIIδ and RyR inhibition, SN treatment decreased Ca2+ spark frequency and dimensions in cardiomyocytes during isoproterenol challenge, and reduced the incidence of Ca2+ waves, delayed afterdepolarizations, and spontaneous action potentials. SN treatment also lowered the incidence of early afterdepolarizations during isoproterenol; an effect paralleled by reduced magnitude of L-type Ca2+ current. CONCLUSIONS: SN production is upregulated in conditions with cardiomyocyte Ca2+ dysregulation and offers compensatory protection against cardiomyocyte mechanisms of arrhythmia, which may underlie its putative use as a biomarker in at-risk patients.

Calmodulin inhibition of human RyR2 channels requires phosphorylation of RyR2-S2808 or RyR2-S2814.

Calmodulin (CaM) is a Ca-binding protein that binds to, and can directly inhibit cardiac ryanodine receptor calcium release channels (RyR2). Animal studies have shown that RyR2 hyperphosphorylation reduces CaM binding to RyR2 in failing hearts, but data are lacking on how CaM regulates human RyR2 and how this regulation is affected by RyR2 phosphorylation. Physiological concentrations of CaM (100 nM) inhibited the diastolic activity of RyR2 isolated from failing human hearts by ~50% but had no effect on RyR2 from healthy human hearts. Using FRET between donor-FKBP12.6 and acceptor-CaM bound to RyR2, we determined that CaM binds to RyR2 from healthy human heart with a Kd = 121 ±â€¯14 nM. Ex-vivo phosphorylation/dephosphorylation experiments suggested that the divergent CaM regulation of healthy and failing human RyR2 was caused by differences in RyR2 phosphorylation by protein kinase A and Ca-CaM-dependent kinase II. Ca2+-spark measurements in murine cardiomyocytes harbouring RyR2 phosphomimetic or phosphoablated mutants at S2814 and S2808 suggest that phosphorylation of residues corresponding to either human RyR2-S2808 or S2814 is both necessary and sufficient for RyR2 regulation by CaM. Our results challenge the current concept that CaM universally functions as a canonical inhibitor of RyR2 across species. Rather, CaM's biological action on human RyR2 appears to be more nuanced, with inhibitory activity only on phosphorylated RyR2 channels, which occurs during exercise or in patients with heart failure.

Cardiac Calcium Release Channel (Ryanodine Receptor 2) Regulation by Halogenated Anesthetics.

BACKGROUND: Halogenated anesthetics activate cardiac ryanodine receptor 2-mediated sarcoplasmic reticulum Ca release, leading to sarcoplasmic reticulum Ca depletion, reduced cardiac function, and providing cell protection against ischemia-reperfusion injury. Anesthetic activation of ryanodine receptor 2 is poorly defined, leaving aspects of the protective mechanism uncertain. METHODS: Ryanodine receptor 2 from the sheep heart was incorporated into artificial lipid bilayers, and their gating properties were measured in response to five halogenated anesthetics. RESULTS: Each anesthetic rapidly and reversibly activated ryanodine receptor 2, but only from the cytoplasmic side. Relative activation levels were as follows: halothane (approximately 4-fold; n = 8), desflurane and enflurane (approximately 3-fold,n = 9), and isoflurane and sevoflurane (approximately 1.5-fold, n = 7, 10). Half-activating concentrations (Ka) were in the range 1.3 to 2.1 mM (1.4 to 2.6 minimum alveolar concentration [MAC]) with the exception of isoflurane (5.3 mM, 6.6 minimum alveolar concentration). Dantrolene (10 µM with 100 nM calmodulin) inhibited ryanodine receptor 2 by 40% but did not alter the Ka for halothane activation. Halothane potentiated luminal and cytoplasmic Ca activation of ryanodine receptor 2 but had no effect on Mg inhibition. Halothane activated ryanodine receptor 2 in the absence and presence (2 mM) of adenosine triphosphate (ATP). Adenosine, a competitive antagonist to ATP activation of ryanodine receptor 2, did not antagonize halothane activation in the absence of ATP. CONCLUSIONS: At clinical concentrations (1 MAC), halothane desflurane and enflurane activated ryanodine receptor 2, whereas isoflurane and sevoflurane were ineffective. Dantrolene inhibition of ryanodine receptor 2 substantially negated the activating effects of anesthetics. Halothane acted independently of the adenine nucleotide-binding site on ryanodine receptor 2. The previously observed adenosine antagonism of halothane activation of sarcoplasmic reticulum Ca release was due to competition between adenosine and ATP, rather than between halothane and ATP.

Essential Role of Calmodulin in RyR Inhibition by Dantrolene.

Dantrolene is the first line therapy of malignant hyperthermia. Animal studies suggest that dantrolene also protects against heart failure and arrhythmias caused by spontaneous Ca(2+) release. Although dantrolene inhibits Ca(2+) release from the sarcoplasmic reticulum of skeletal and cardiac muscle preparations, its mechanism of action has remained controversial, because dantrolene does not inhibit single ryanodine receptor (RyR) Ca(2+) release channels in lipid bilayers. Here we test the hypothesis that calmodulin (CaM), a physiologic RyR binding partner that is lost during incorporation into lipid bilayers, is required for dantrolene inhibition of RyR channels. In single channel recordings (100 nM cytoplasmic [Ca(2+)] + 2 mM ATP), dantrolene caused inhibition of RyR1 (rabbit skeletal muscle) and RyR2 (sheep) with a maximal inhibition of Po (Emax) to 52 ± 4% of control only after adding physiologic [CaM] = 100 nM. Dantrolene inhibited RyR2 with an IC50 of 0.16 ± 0.03 µM. Mutant N98S-CaM facilitated dantrolene inhibition with an IC50 = 5.9 ± 0.3 nM. In mouse cardiomyocytes, dantrolene had no effect on cardiac Ca(2+) release in the absence of CaM, but reduced Ca(2+) wave frequency (IC50 = 0.42 ± 0.18 µM, Emax = 47 ± 4%) and amplitude (IC50 = 0.19 ± 0.04 µM, Emax = 66 ± 4%) in the presence of 100 nM CaM. We conclude that CaM is essential for dantrolene inhibition of RyR1 and RyR2. Its absence explains why dantrolene inhibition of single RyR channels has not been previously observed.

A domain peptide of the cardiac ryanodine receptor regulates channel sensitivity to luminal Ca2+ via cytoplasmic Ca2+ sites.

The clustering of cardiac RyR mutations, linked to sudden cardiac death (SCD), into several regions in the amino acid sequence underlies the hypothesis that these mutations interfere with stabilising interactions between different domains of the RyR2. SCD mutations cause increased channel sensitivity to cytoplasmic and luminal Ca(2+). A synthetic peptide corresponding to part of the central domain (DPc10:(2460)G-P(2495)) was designed to destabilise the interaction of the N-terminal and central domains of wild-type RyR2 and mimic the effects of SCD mutations. With Ca(2+) as the sole regulating ion, DPc10 caused increased channel activity which could be reversed by removal of the peptide whereas in the presence of ATP DPc10 caused no activation. In support of the domain destablising hypothesis, the corresponding peptide (DPc10-mut) containing the CPVT mutation R2474S did not affect channel activity under any circumstances. DPc10-induced activation was due to a small increase in RyR2 sensitivity to cytoplasmic Ca(2+) and a large increase in the magnitude of luminal Ca(2+) activation. The increase in the luminal Ca(2+) response appeared reliant on the luminal-to-cytoplasmic Ca(2+) flux in the channel, indicating that luminal Ca(2+) was activating the RyR2 via its cytoplasmic Ca(2+) sites. DPc10 had no significant effect on the RyR2 gating associated with luminal Ca(2+) sensing sites. The results were fitted by the luminal-triggered Ca(2+) feed-through model and the effects of DPc10 were explained entirely by perturbations in cytoplasmic Ca(2+)-activation mechanism.

Ca2+ stores regulate ryanodine receptor Ca2+ release channels via luminal and cytosolic Ca2+ sites.

1. In muscle, intracellular calcium concentration, hence skeletal muscle force and cardiac output, is regulated by uptake and release of calcium from the sarcoplasmic reticulum. The ryanodine receptor (RyR) forms the calcium release channel in the sarcoplasmic reticulum. 2. The free [Ca2+] in the sarcoplasmic reticulum regulates the excitability of this store by stimulating the Ca2+ release channels in its membrane. This process involves Ca2+-sensing mechanisms on both the luminal and cytoplasmic sides of the RyR. In the cardiac RyR, these have been shown to be a luminal Ca2+ activation site (L-site; 60 micromol/L affinity), a cytoplasmic activation site (A-site; 0.9 micromol/L affinity) and a cytoplasmic Ca2+ inactivation site (I2-site; 1.2 micromol/L affinity). 3. Cardiac RyR activation by luminal Ca2+ occurs by a multistep process dubbed 'luminal-triggered Ca2+ feed-through'. Binding of Ca2+ to the L-site initiates brief (1 msec) openings at a rate of up to 10/s. Once the pore is open, luminal Ca2+ has access to the A-site (producing up to 30-fold prolongation of openings) and to the I2-site (causing inactivation at high levels of Ca2+ feed-through). 4. The present paper reviews the evidence for the principal aspects of the 'luminal-triggered Ca2+ feed-through' model, the properties of the various Ca2+-dependent gating mechanisms and their likely role in controlling sarcoplasmic reticulum (SR) Ca2+ release in cardiac muscle. 5. The model makes the following important predictions: (i) there will be a close link between luminal and cytoplasmic regulation of RyRs and any cofactor that prolongs channel openings triggered by cytoplasmic Ca2+ will also promote RyR activation by luminal Ca2+; (ii) luminal Mg2+ (1 mmol/L) is essential for the control of SR excitability in cardiac muscle by luminal Ca2+; and (iii) the different RyR isoforms in skeletal and cardiac muscle will be controlled quite differently by the luminal milieu. For example, Mg2+ in the SR lumen (approximately 1 mmol/L) can strongly inhibit RyR2 by competing with Ca2+ for the L-site, whereas RyR1 is not affected by luminal Mg2+.

Coupled calcium release channels and their regulation by luminal and cytosolic ions.

Contraction in skeletal and cardiac muscle occurs when Ca(2+) is released from the sarcoplasmic reticulum (SR) through ryanodine receptor (RyR) Ca(2+) release channels. Several isoforms of the RyR exist throughout the animal kingdom, which are modulated by ATP, Ca(2+) and Mg(2+) in the cytoplasm and by Ca(2+) in the lumen of the SR. This review brings to light recent findings on their mechanisms of action in the mammalian isoforms RyR-1 and RyR-2 with an emphasis on RyR-1 from skeletal muscle. Cytoplasmic Mg(2+) is a potent RyR antagonist that binds to two classes of cytoplasmic site, identified as low-affinity, non-specific inhibition sites and high-affinity Ca(2+) activation sites (A-sites). Mg(2+) inhibition at the A-sites is very sensitive to the cytoplasmic and luminal milieu. Cytoplasmic Ca(2+), Mg(2+) and monovalent cations compete for the A-sites. In isolated RyRs, luminal Ca(2+) alters the Mg(2+) affinity of the A-site by an allosteric mechanism mediated by luminal sites. However, in close-packed RyR arrays luminal Ca(2+) can also compete with cytoplasmic ions for the A-site. Activation of RyRs by luminal Ca(2+) has been attributed to either Ca(2+) feedthrough to A-sites or to Ca(2+) regulatory sites on the luminal side of the RyR. As yet there is no consensus on just how luminal Ca(2+) alters RyR activation. Recent evidence indicates that both mechanisms operate and are likely to be important. Allosteric regulation of A-site Mg(2+) affinity could trigger Ca(2+) release, which is reinforced by Ca(2+) feedthrough.

Regulation of ryanodine receptors by calsequestrin: effect of high luminal Ca2+ and phosphorylation.

Calsequestrin, the major calcium sequestering protein in the sarcoplasmic reticulum of muscle, forms a quaternary complex with the ryanodine receptor calcium release channel and the intrinsic membrane proteins triadin and junctin. We have investigated the possibility that calsequestrin is a luminal calcium concentration sensor for the ryanodine receptor. We measured the luminal calcium concentration at which calsequestrin dissociates from the ryanodine receptor and the effect of calsequestrin on the response of the ryanodine receptor to changes in luminal calcium. We provide electrophysiological and biochemical evidence that: 1), luminal calcium concentration of >/=4 mM dissociates calsequestrin from junctional face membrane, whereas in the range of 1-3 mM calsequestrin remains attached; 2), the association with calsequestrin inhibits ryanodine receptor activity, but amplifies its response to changes in luminal calcium concentration; and 3), under physiological calcium conditions (1 mM), phosphorylation of calsequestrin does not alter its ability to inhibit native ryanodine receptor activity when the anchoring proteins triadin and junctin are present. These data suggest that the quaternary complex is intact in vivo, and provides further evidence that calsequestrin is involved in the sarcoplasmic reticulum calcium signaling pathway and has a role as a luminal calcium sensor for the ryanodine receptor.

Regulation of skeletal ryanodine receptors by dihydropyridine receptor II-III loop C-region peptides: relief of Mg2+ inhibition.

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 a1 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 (>10 microM), 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.

The random-coil 'C' fragment of the dihydropyridine receptor II-III loop can activate or inhibit native skeletal ryanodine receptors.

The actions of peptide C, corresponding to (724)Glu-Pro(760) 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 microM. 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 microM). 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 x 10(5) s(-1).M(-1) was obtained for activation and a lower estimate for the rate constant for inhibition of 5.9 x 10(3) s(-1).M(-1). The combined actions of peptide C and peptide A (II-III loop sequence (671)Thr-Leu(690)) 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.

Calsequestrin is an inhibitor of skeletal muscle ryanodine receptor calcium release channels.

We provide novel evidence that the sarcoplasmic reticulum calcium binding protein, calsequestrin, inhibits native ryanodine receptor calcium release channel activity. Calsequestrin dissociation from junctional face membrane was achieved by increasing luminal (trans) ionic strength from 250 to 500 mM with CsCl or by exposing the luminal side of ryanodine receptors to high [Ca(2+)] (13 mM) and dissociation was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Calsequestrin dissociation caused a 10-fold increase in the duration of ryanodine receptor channel opening in lipid bilayers. Adding calsequestrin back to the luminal side of the channel after dissociation reversed this increased activity. In addition, an anticalsequestrin antibody added to the luminal solution reduced ryanodine receptor activity before, but not after, calsequestrin dissociation. A population of ryanodine receptors (approximately 35%) may have initially lacked calsequestrin, because their activity was high and was unaffected by increasing ionic strength or by anticalsequestrin antibody: their activity fell when purified calsequestrin was added and they then responded to antibody. In contrast to native ryanodine receptors, purified channels, depleted of triadin and calsequestrin, were not inhibited by calsequestrin. We suggest that calsequestrin reduces ryanodine receptor activity by binding to a coprotein, possibly to the luminal domain of triadin.