Ca2+-Dependent Phosphorylation of RyR2 Can Uncouple Channel Gating from Direct Cytosolic Ca2+ Regulation

Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca2+ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca2+. We show that hyperphosphorylation by exogenous PKA increases open probability (Po) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca2+; lowering [Ca2+] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca2+-sensitive mode of gating. We additionally found that cytosolic incubation with Mg2+/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca2+. We show that the endogenous kinase is both Ca2+-dependent and sensitive to inhibitors of PKC. Moreover, the Ca2+-dependent, endogenous kinase–induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca2+-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca2+ regulation

FKBP12 Activates the Cardiac Ryanodine Receptor Ca2+-Release Channel and Is Antagonised by FKBP12.6

Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca2+-homeostasis in heart failure but there is controversy over the functional effects of FKBP12.6 on RyR2 channel gating. We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca2+-induced Ca2+-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca2+, whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 µM) increased Ca2+-wave frequency and decreased the SR Ca2+-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12

Identifikasi Karakter Siswa Menggunakan Metode K-Means (Studi Kasus Sdn 156 Pekanbaru)

Good character education can have a characteristic impact on students. each student has a different character. Various ways done by the school in character education based on kemendiknas, including State Elementary School 156 Pekanbaru. Problems that arise in the field is there is no method that can determine the character of the students so that the school's special teachers can not understand precisely the characters in the students. The lack of understanding of the character of the students makes the vision of the school mission has not been seen so that character education in SDN 156 Pekanbaru has not been right target. Therefore, it needs to be done grouping student character in SDN 156 Pekanbaru with the aim of school know character owned by students in school. The K-Means algorithm is used to classify the character of the students with the number of clusters as much as 2 using the six attributes of characters studied: Honest, disciplined, confident, caring, creative and responsible with 130 student data. The results of K-Means manual calculation with sample data 10 data from 130 data that is weak character (C1) amounted to 1 student and weak character of 9 students, this result is same with calculation executed by RapidMiner application. Test results with 130 data using RapidMiner resulted in the number of students with weak character 26 students with the average centroid (0.665) with caring and creative characters. While students who have strong character 104 students with average value of centroid (0.900) with honest character, discipline, confidence, and responsibility. The result of character grouping based on class cluster position in RapidMiner is grade 3 which has weak character (C1) 8 students from 35 students, grade 4 is 8 out of 24 students, 5th grade is 1 of 17 students and grade 6 is 9 of 46 students. While clusters with strong characters (C2) class 3 amounted to 27 students, grade 4 amounted to 24 students, class 5 amounted to 16 students, and grade 6 amounted to 37 students. From the results of this study is expected Strong characters can be developed by school continue to perform habits which involves the students so that the characters in the students can be seen while for the caring and creative characters so as not to be weak then the school always provide guidance to the students and give examples of good habits and activities that can be followed by students in school

EHMTI-0026. Neuroprolotherapy and acupuncture for clinical trial of acute and chronic migraine treatment

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) are Ca2+-mobilizing messengers important for modulating cardiac excitation–contraction coupling and pathophysiology. CD38, which belongs to the ADP-ribosyl cyclase family, catalyzes synthesis of both NAADP and cADPR in vitro. However, it remains unclear whether this is the main enzyme for their production under physiological conditions. Here we show that membrane fractions from WT but not CD38−/− mouse hearts supported NAADP and cADPR synthesis. Membrane permeabilization of cardiac myocytes with saponin and/or Triton X-100 increased NAADP synthesis, indicating that intracellular CD38 contributes to NAADP production. The permeabilization also permitted immunostaining of CD38, with a striated pattern in WT myocytes, whereas CD38−/− myocytes and nonpermeabilized WT myocytes showed little or no staining, without striation. A component of β-adrenoreceptor signaling in the heart involves NAADP and lysosomes. Accordingly, in the presence of isoproterenol, Ca2+ transients and contraction amplitudes were smaller in CD38−/− myocytes than in the WT. In addition, suppressing lysosomal function with bafilomycin A1 reduced the isoproterenol-induced increase in Ca2+ transients in cardiac myocytes from WT but not CD38−/− mice. Whole hearts isolated from CD38−/− mice and exposed to isoproterenol showed reduced arrhythmias. SAN4825, an ADP-ribosyl cyclase inhibitor that reduces cADPR and NAADP synthesis in mouse membrane fractions, was shown to bind to CD38 in docking simulations and reduced the isoproterenol-induced arrhythmias in WT hearts. These observations support generation of NAADP and cADPR by intracellular CD38, which contributes to effects of β-adrenoreceptor stimulation to increase both Ca2+ transients and the tendency to disturb heart rhythm

Flux regulation of cardiac ryanodine receptor channels

The cardiac type 2 ryanodine receptor (RYR2) is activated by Ca2+-induced Ca2+ release (CICR). The inherent positive feedback of CICR is well controlled in cells, but the nature of this control is debated. Here, we explore how the Ca2+ flux (lumen-to-cytosol) carried by an open RYR2 channel influences its own cytosolic Ca2+ regulatory sites as well as those on a neighboring channel. Both flux-dependent activation and inhibition of single channels were detected when there were super-physiological Ca2+ fluxes (>3 pA). Single-channel results indicate a pore inhibition site distance of 1.2 ± 0.16 nm and that the activation site on an open channel is shielded/protected from its own flux. Our results indicate that the Ca2+ flux mediated by an open RYR2 channel in cells (∼0.5 pA) is too small to substantially regulate (activate or inhibit) the channel carrying it, even though it is sufficient to activate a neighboring RYR2 channel

Role of Ca2+ in the rapid cooling-induced Ca2+ release from sarcoplasmic reticulum in ferret cardiac muscles

Rapid lowering of the solution temperature (rapid cooling, RC) from 24 to 3°C within 3 s releases considerable amounts of Ca2+ from the sarcoplasmic reticulum (SR) in mammalian cardiac muscles. In this study, we investigated the intracellular mechanism of RC-induced Ca2+ release, especially the role of Ca2+, in ferret ventricular muscle. Saponin-treated skinned trabeculae were placed in a glass capillary, and the amount of Ca2+ released from the SR by RC and caffeine (50 mM) was measured with fluo-3. It was estimated that in the presence of ATP about 45% of the Ca2+ content in the SR was released by RC. The amount of SR Ca2+ released by RC was unchanged by the replacement of ATP by AMP-PCP (a non-hydrolysable ATP analogue and agonist for the ryanodine receptor but not for the Ca2+ pump of SR), suggesting that the suppression of the Ca2+ pump of SR at low temperature might not be a major mechanism in RC-induced Ca2+ release. The free Ca2+ concentration of the solution used for triggering RC-induced Ca2+ release was estimated to be only about 20 nM with fluo-3 or aequorin. When this solution was applied to the preparation at 3°C, only a small amount of Ca2+ was released from SR presumably by the Ca2+-induced Ca2+ release (CICR) mechanism. Thus, in mammalian cardiac muscles, RC releases a part of the (<50%) stored Ca2+ contained in the SR, and the mechanism of RC-induced Ca2+ release may differ from that of CICR, which is thought to play a role in frog skeletal muscle fibres that express ryanodine receptors of different types

Simvastatin activates single skeletal RyR1 channels but exerts more complex regulation of the cardiac RyR2 isoform

Background and Purpose: Statins are amongst the most widely prescribed drugs for those at risk of cardiovascular disease, lowering cholesterol levels by inhibiting 3‐hydroxy‐3‐methylglutaryl (HMG)‐CoA reductase. Although effective at preventing cardiovascular disease, statin use is associated with muscle weakness, myopathies and, occasionally, fatal rhabdomyolysis. As simvastatin, a commonly prescribed statin, promotes Ca²⁺ release from sarcoplasmic reticulum (SR) vesicles, we investigated if simvastatin directly activates skeletal (RyR1) and cardiac (RyR2) ryanodine receptors. Experimental Approach: RyR1 and RyR2 single‐channel behaviour was investigated after incorporation of sheep cardiac or mouse skeletal SR into planar phospholipid bilayers under voltage‐clamp conditions. LC‐MS was used to monitor the kinetics of interconversion of simvastatin between hydroxy‐acid and lactone forms during these experiments. Cardiac and skeletal myocytes were permeabilised to examine simvastatin modulation of SR Ca²⁺ release. Key Results: Hydroxy acid simvastatin (active at HMG‐CoA reductase) significantly and reversibly increased RyR1 open probability (Po) and shifted the distribution of Ca²⁺ spark frequency towards higher values in skeletal fibres. In contrast, simvastatin reduced RyR2 Po and shifted the distribution of spark frequency towards lower values in ventricular cardiomyocytes. The lactone pro‐drug form of simvastatin (inactive at HMG‐CoA reductase) also activated RyR1, suggesting that the HMG‐CoA inhibitor pharmacophore was not responsible for RyR1 activation. Conclusion and Implications: Simvastatin interacts with RyR1 to increase SR Ca²⁺ release and thus may contribute to its reported adverse effects on skeletal muscle. The ability of low concentrations of simvastatin to reduce RyR2 Po may also protect against Ca²⁺‐dependent arrhythmias and sudden cardiac death

The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity

Unambiguous observation of blocked states reveals altered, blocker-induced, cardiac ryanodine receptor gating

The flow of ions through membrane channels is precisely regulated by gates. The architecture and function of these elements have been studied extensively, shedding light on the mechanisms underlying gating. Recent investigations have focused on ion occupancy of the channel’s selectivity filter and its ability to alter gating, with most studies involving prokaryotic K+ channels. Some studies used large quaternary ammonium blocker molecules to examine the effects of altered ionic flux on gating. However, the absence of blocking events that are visibly distinct from closing events in K+ channels makes unambiguous interpretation of data from single channel recordings difficult. In this study, the large K+ conductance of the RyR2 channel permits direct observation of blocking events as distinct subconductance states and for the first time demonstrates the differential effects of blocker molecules on channel gating. This experimental platform provides valuable insights into mechanisms of blocker-induced modulation of ion channel gating

Selective inhibitors of cardiac ADPR cyclase as novel anti-arrhythmic compounds

ADP-ribosyl cyclases (ADPRCs) catalyse the conversion of nicotinamide adenine dinucleotide to cyclic adenosine diphosphoribose (cADPR) which is a second messenger involved in Ca2+ mobilisation from intracellular stores. Via its interaction with the ryanodine receptor Ca2+ channel in the heart, cADPR may exert arrhythmogenic activity. To test this hypothesis, we have studied the effect of novel cardiac ADPRC inhibitors in vitro and in vivo in models of ventricular arrhythmias. Using a high-throughput screening approach on cardiac sarcoplasmic reticulum membranes isolated from pig and rat and nicotinamide hypoxanthine dinuleotide as a surrogate substrate, we have identified potent and selective inhibitors of an intracellular, membrane-bound cardiac ADPRC that are different from the two known mammalian ADPRCs, CD38 and CD157/Bst1. We show that two structurally distinct cardiac ADPRC inhibitors, SAN2589 and SAN4825, prevent the formation of spontaneous action potentials in guinea pig papillary muscle in vitro and that compound SAN4825 is active in vivo in delaying ventricular fibrillation and cardiac arrest in a guinea pig model of Ca2+ overload-induced arrhythmia. Inhibition of cardiac ADPRC prevents Ca2+ overload-induced spontaneous depolarizations and ventricular fibrillation and may thus provide a novel therapeutic principle for the treatment of cardiac arrhythmias