http://nova.newcastle.edu.au/vital/access/services/Feed ${session.getAttribute("locale")} 5 http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:2744 The free [Ca²⁺] in endoplasmic/sarcoplasmic reticulum Ca²⁺ stores regulates excitability of Ca²⁺ release by stimulating the Ca²⁺ release channels. Just how the stored Ca²⁺ regulates activation of these channels is still disputed. One proposal attributes luminal Ca²⁺-activation to luminal facing regulatory sites, whereas another envisages Ca²⁺ permeation to cytoplasmic sites. This study develops a unified model for luminal Ca²⁺ activation for single cardiac ryanodine receptors (RyR₂) and RyRs in coupled clusters in artificial lipid bilayers. It is shown that luminal regulation of RyR₂ involves three modes of action associated with Ca²⁺ sensors in different parts of the molecule; a luminal activation site (L-site, 60 μM affinity), a cytoplasmic activation site (A-site, 0.9 μM affinity), and a novel cytoplasmic inactivation site (I₂-site, 1.2 μM affinity). RyR activation by luminal Ca²⁺ is demonstrated to occur by a multistep process dubbed luminal-triggered Ca²⁺ feedthrough. Ca²⁺ binding to the L-site initiates brief openings (1 ms duration at 1–10 s⁻¹) allowing luminal Ca²⁺ to access the A-site, producing up to 30-fold prolongation of openings. The model explains a broad data set, reconciles previous conflicting observations and provides a foundation for understanding the action of pharmacological agents, RyR-associated proteins, and RyR₂ mutations on a range of Ca²⁺-mediated physiological and pathological processes. 2012-05-29T02:16:02.803Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:34 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 >= 4mM 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. 2012-03-12T07:09:09.726Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7011 The ryanodine receptors form the calcium release channel in the membrane of the sarcoplasmic reticulum (SR, the main intracellular Ca²⁺ store). The importance of ryanodine receptors (RyRs) to cardiac pacemaking and rhythmicity is highlighted by more than 69 mutations, RyR mutations, which underlie arrhythmias and sudden cardiac death. Although most of these mutations lie in cytoplasmic domains, they all cause increased RyR activation by Ca²⁺ in the SR lumen. Presented here is a review of the mechanisms by which cytoplasmic domains of the RyR can determine luminal activation. 2012-01-30T05:05:52.317Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:225 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 ((725)Phe-Pro(742)) of the linker between the 2nd and 3rd repeats (II-III loop) of the a, 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 RyR 1 channels in lipid bilayers with three key RyR regulators, Ca2+, Mg2+ and ATP. We identified four discrete actions: two novel, low-affinity (> 10 mu M), 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. 2010-04-27T06:00:45.900Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:4426 In cardiac muscle, intracellular Ca²⁺ and Mg²⁺ are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [Ca²⁺] in the SR ([Ca²⁺]L) stimulates the Ca²⁺ release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal Mg²⁺, which has not been regarded as an important regulator of Ca²⁺ release. The effects of luminal Ca²⁺ and Mg²⁺ on sheep RYR2 were measured in lipid bilayers. Cytoplasmic and luminal Ca²⁺ produced a synergistic increase in the opening rate of RYRs. A novel, high affinity inhibition of RYR2 by luminal Mg²⁺ was observed, pointing to an important physiological role for luminal Mg²⁺ in cardiac muscle. At diastolic [Ca²⁺]C, luminal Mg²⁺ inhibition was voltage independent, with Ki=45 μM at luminal [Ca²⁺] ([Ca²⁺]L) = 100 μM. Luminal and cytoplasmic Mg²⁺ inhibition was alleviated by increasing [Ca²⁺]L or [Ca²⁺]C. Ca²⁺ and Mg²⁺ on opposite sides of the bilayer exhibited competitive effects on RYRs, indicating that they can compete via the pore for common sites. The data were accurately fitted by a model based on a tetrameric RYR structure with four Ca²⁺-sensing mechanisms on each subunit: activating luminal L-site (40-μM affi nity for Mg²⁺ and Ca²⁺), cytoplasmic A-site (1.2 μM for Ca²⁺ and 60 μM for Mg²⁺), inactivating cytoplasmic I₁-site (~10 mM for Ca²⁺ and Mg²⁺), and I₂-site (1.2 μM for Ca²⁺). Activation of three or more subunits will cause channel opening. Mg²⁺ inhibition occurs primarily by Mg²⁺ displacing Ca²⁺ from the L- and A-sites, and Mg²⁺ fails to open the channel. The model predicts that under physiological conditions, SR load–dependent Ca²⁺ release (1) is mainly determined by Ca²⁺ displacement of Mg²⁺ from the L-site as SR loading increases, and (2) depends on the properties of both luminal and cytoplasmic activation mechanisms. 2010-04-27T04:54:19.479Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:5775 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 μmol/L affinity), a cytoplasmic activation site (A-site; 0.9 μmol/L affinity) and a cytoplasmic Ca²⁺ inactivation site (I₂-site; 1.2 μmol/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⁺. 2010-04-27T04:50:27.054Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:5571 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 Ca²⁺. A synthetic peptide corresponding to part of the central domain (DPc10:²⁴⁶⁰ G-P²⁴⁹⁵) 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 Ca²⁺ 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 Ca²⁺ and a large increase in the magnitude of luminal Ca²⁺ activation. The increase in the luminal Ca²⁺ response appeared reliant on the luminal-to-cytoplasmic Ca²⁺ flux in the channel, indicating that luminal Ca²⁺ was activating the RyR2 via its cytoplasmic Ca²⁺ sites. DPc10 had no significant effect on the RyR2 gating associated with luminal Ca2+ sensing sites. The results were fitted by the luminal-triggered Ca²⁺ feed-through model and the effects of DPc10 were explained entirely by perturbations in cytoplasmic Ca²⁺ -activation mechanism. 2010-04-27T04:40:01.020Z ]]>