Cardiac Sarcoplasmic Reticulum Vesicles Research Articles

Junctophilin-2 allosterically interacts with ryanodine receptor type 2 to regulate calcium release units in mouse cardiomyocytes.

The interaction between junctophilin-2 (JPH2) and ryanodine receptor type 2 (RyR2) regulated Ca2+ signaling in mouse cardiomyocytes. However, their exact interaction remains unclear. This study elucidates the interaction between JPH2 with RyR2 using co-immunoprecipitation of cardiac sarcoplasmic reticulum vesicles. Additionally, a glutathione S-transferase (GST) pull-down analysis was performed to investigate the physical interaction between RyR2 and JPH2 fragments. JPH2 interacted with RyR2 and the C terminus of the JPH2 protein can pull-down RyR2 receptors. Confocal immunofluorescence imaging indicated that the majority of JPH2 and RyR2 proteins were colocalized near Z-lines in isolated mouse cardiomyocytes. Knockdown of JPH2 reduced the amplitude of Ca2+ transients and disrupted its interaction with RyR2. Therefore, the C-terminus domain of JPH2 is required for interactions with RyR2 in mouse cardiomyocytes, which provides a molecular mechanism for seeking Ca2+-related disease prevention strategies.

New N-aryl-N-alkyl-thiophene-2-carboxamide compound enhances intracellular Ca2+ dynamics by increasing SERCA2a Ca2+ pumping

The type 2a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a central role in the intracellular Ca2+ homeostasis of cardiac myocytes, pumping Ca2+ from the cytoplasm into the sarcoplasmic reticulum (SR) lumen to maintain relaxation (diastole) and prepare for contraction (systole). Diminished SERCA2a function has been reported in several pathological conditions, including heart failure. Therefore, development of new drugs that improve SERCA2a Ca2+ transport is of great clinical significance. In this study, we characterized the effect of a recently identified N-aryl-N-alkyl-thiophene-2-carboxamide (or compound 1) on SERCA2a Ca2+-ATPase and Ca2+ transport activities in cardiac SR vesicles, and on Ca2+ regulation in a HEK293 cell expression system and in mouse ventricular myocytes. We found that compound 1 enhances SERCA2a Ca2+-ATPase and Ca2+ transport in SR vesicles. Fluorescence lifetime measurements of fluorescence resonance energy transfer between SERCA2a and phospholamban indicated that compound 1 interacts with the SERCA-phospholamban complex. Measurement of endoplasmic reticulum Ca2+ dynamics in HEK293 cells expressing human SERCA2a showed that compound 1 increases endoplasmic reticulum Ca2+ load by enhancing SERCA2a-mediated Ca2+ transport. Analysis of cytosolic Ca2+ dynamics in mouse ventricular myocytes revealed that compound 1 increases the action potential-induced Ca2+ transients and SR Ca2+ load, with negligible effects on L-type Ca2+ channels and Na+/Ca2+ exchanger. However, during adrenergic receptor activation, compound 1 did not further increase Ca2+ transients and SR Ca2+ load, but it decreased the propensity toward Ca2+ waves. Suggestive of concurrent desirable effects of compound 1 on RyR2, [3H]-ryanodine binding to cardiac SR vesicles shows a small decrease in nM Ca2+ and a small increase in μM Ca2+. Accordingly, compound 1 slightly decreased Ca2+ sparks in permeabilized myocytes. Thus, this novel compound shows promising characteristics to improve intracellular Ca2+ dynamics in cardiomyocytes that exhibit reduced SERCA2a Ca2+ uptake, as found in failing hearts.

Dysregulated Zn2+ homeostasis impairs cardiac type-2 ryanodine receptor and mitsugumin 23 functions, leading to sarcoplasmic reticulum Ca2+ leakage

Aberrant Zn2+ homeostasis is associated with dysregulated intracellular Ca2+ release, resulting in chronic heart failure. In the failing heart a small population of cardiac ryanodine receptors (RyR2) displays sub-conductance-state gating leading to Ca2+ leakage from sarcoplasmic reticulum (SR) stores, which impairs cardiac contractility. Previous evidence suggests contribution of RyR2-independent Ca2+ leakage through an uncharacterized mechanism. We sought to examine the role of Zn2+ in shaping intracellular Ca2+ release in cardiac muscle. Cardiac SR vesicles prepared from sheep or mouse ventricular tissue were incorporated into phospholipid bilayers under voltage-clamp conditions, and the direct action of Zn2+ on RyR2 channel function was examined. Under diastolic conditions, the addition of pathophysiological concentrations of Zn2+ (≥2 nm) caused dysregulated RyR2-channel openings. Our data also revealed that RyR2 channels are not the only SR Ca2+-permeable channels regulated by Zn2+. Elevating the cytosolic Zn2+ concentration to 1 nm increased the activity of the transmembrane protein mitsugumin 23 (MG23). The current amplitude of the MG23 full-open state was consistent with that previously reported for RyR2 sub-conductance gating, suggesting that in heart failure in which Zn2+ levels are elevated, RyR2 channels do not gate in a sub-conductance state, but rather MG23-gating becomes more apparent. We also show that in H9C2 cells exposed to ischemic conditions, intracellular Zn2+ levels are elevated, coinciding with increased MG23 expression. In conclusion, these data suggest that dysregulated Zn2+ homeostasis alters the function of both RyR2 and MG23 and that both ion channels play a key role in diastolic SR Ca2+ leakage.

A pyridone derivative activates SERCA2a by attenuating the inhibitory effect of phospholamban

The cardiac sarco/endoplasmic reticulum Ca2+-dependent ATPase 2a (SERCA2a) plays a central role in Ca2+ handling within cardiomyocytes and is negatively regulated by phospholamban (PLN), a sarcoplasmic reticulum (SR) membrane protein. The activation of SERCA2a, which has been reported to improve cardiac dysfunction in heart failure, is a potential therapeutic approach for heart failure. Therefore, we developed a novel small molecule, compound A and characterized it both in vitro and in vivo. Compound A activated the Ca2+-dependent ATPase activity of cardiac SR vesicles but not that of skeletal muscle SR vesicles that lack PLN. The surface plasmon resonance assay revealed a direct interaction between compound A and PLN, suggesting that the binding of compound A to PLN attenuates its inhibition of SERCA2a, resulting in SERCA2a activation. This was substantiated by inhibition of the compound A-mediated increase in Ca2+ levels within the SR of HL-1 cells by thapsigargin, a SERCA inhibitor. Compound A also increased the Ca2+ transients and contraction and relaxation of isolated adult rat cardiomyocytes. In isolated perfused rat hearts, the compound A enhanced systolic and diastolic functions. Further, an infusion of compound A (30mg/kg, i.v. bolus followed by 2mg/kg/min, i.v. infusion) significantly enhanced the diastolic function in anesthetized normal rats. These results indicate that compound A is a novel SERCA2a activator, which attenuates PLN inhibition and enhances the systolic and diastolic functions of the heart in vitro and in vivo. Therefore, compound A might be a novel therapeutic lead for heart failure.

Istaroxime stimulates SERCA2a and accelerates calcium cycling in heart failure by relieving phospholamban inhibition

Calcium handling is known to be deranged in heart failure. Interventions aimed at improving cell Ca(2) (+) cycling may represent a promising approach to heart failure therapy. Istaroxime is a new luso-inotropic compound that stimulates cardiac contractility and relaxation in healthy and failing animal models and in patients with acute heart failure (AHF) syndrome. Istaroxime is a Na-K ATPase inhibitor with the unique property of increasing sarcoplasmic reticulum (SR) SERCA2a activity as shown in heart microsomes from humans and guinea pigs. The present study addressed the molecular mechanism by which istaroxime increases SERCA2a activity. To study the effect of istaroxime on SERCA2a-phospholamban (PLB) complex, we applied different methodologies in native dog healthy and failing heart preparations and heterologous canine SERCA2a/PLB co-expressed in Spodoptera frugiperda (Sf21) insect cells. We showed that istaroxime enhances SERCA2a activity, Ca(2) (+) uptake and the Ca(2) (+) -dependent charge movements into dog healthy and failing cardiac SR vesicles. Although not directly demonstrated, the most probable explanation of these activities is the displacement of PLB from SERCA2a.E2 conformation, independently from cAMP/PKA. We propose that this displacement may favour the SERCA2a conformational transition from E2 to E1, thus resulting in the acceleration of Ca(2) (+) cycling. Istaroxime represents the first example of a small molecule that exerts a luso-inotropic effect in the failing human heart through the stimulation of SERCA2a ATPase activity and the enhancement of Ca(2) (+) uptake into the SR by relieving the PLB inhibitory effect on SERCA2a in a cAMP/PKA independent way.

FRET Detection of CaM-RyR2 Binding Modulation by S100A1

Using fluorescence resonance energy transfer (FRET), we are testing the hypothesis that S100A1 competes with calmodulin (CaM) for binding to cardiac ryanodine receptors (RyR2). In isolated pig cardiac sarcoplasmic reticulum (SR) vesicles, we targeted a donor-labeled FKBP (D-FKBP) to the RyR2 cytosolic headpiece. We then detected FRET as a decrease of donor fluorescence in the presence of CaM labeled with acceptor within the N- or C-lobe (denoted AN-CaM and AC-CaM, respectively), thus directly and specifically indexing CaM binding in the proximity of D-FKBP. FRET between D-FKBP and A-CaMs (100 nM) was completely inhibited by unlabeled WT-CaM, with KI ≈ 100 nM, indicating that WT-CaM and A-CaMs compete with similar affinities for the same RyR2 binding site. However, S100A1 (ranging from 0.1 to 30 μM) had no significant effect on FRET when cardiac SR was concurrently incubated with S100A1 and AN-CaM. Upon sequentially incubating the SR with S100A1 first, then with AN-CaM, we found partial inhibition of FRET, but with KI ≈ 30 μM S100A1. This effect is more pronounced in nM Ca2+ (versus μM Ca2+). Intriguingly, FRET between D-FKBP and AC-CaM was not significantly affected by S100A1. S100A1 lowered maximum FRET for AN-CaM but did not significantly change its binding affinity. This suggests that S100A1 allosterically interacts with RyR-CaM binding, rather than directly competing for the same binding site as CaM. We are currently developing a complementary FRET approach, using acceptor-labeled S100A1, to specifically resolve the S100A1 binding to RyR. Ultimately, we aim to elucidate the interplay between S100A1 and CaM binding to RyR.

Epigallocatechin-3-gallate has dual, independent effects on the cardiac sarcoplasmic reticulum/endoplasmic reticulum Ca2+ ATPase

We determined the effects of epigallocatechin-3-gallate (EGCG) and epicatechin (EC), on pump turnover and Ca2+ transport by the cardiac form of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). Fluorescence spectroscopy was used to directly measure SERCA ATPase activity and to measure Ca2+ uptake into cardiac sarcoplasmic reticulum (SR) vesicles and microsomes derived from human embryonic kidney (HEK) cells expressing human cardiac SERCA2a. We found that EGCG reduces the maximum velocity of Ca2+ uptake into cardiac SR vesicles and increases the Ca2+-sensitivity of uptake in a concentration-dependent manner. EC is less potent than EGCG in increasing the Ca2+-sensitivity of uptake and does not affect maximum uptake velocity. The EGCG-dependent reduction in Ca2+ uptake velocity is well correlated with direct inhibition of SERCA. The effect of EGCG on the Ca2+-sensitivity of Ca2+ uptake into cardiac SR vesicles is affected by the phosphorylation status of phospholamban (PLB). When cardiac SERCA2a is expressed in HEK cells without PLB, EGCG reduces the maximum velocity of Ca2+ uptake but does not affect the Ca2+-sensitivity of uptake into microsomes derived from these cells indicating that the effect of EGCG on Ca2+-sensitivity requires the presence of PLB. Our results show that EGCG has dual effects on SERCA function in cardiac SR vesicles: it directly affects SERCA by reducing maximum uptake velocity; it increases the Ca2+-sensitivity of Ca2+ uptake in a manner that appears to depend on the interaction between SERCA and PLB.

Multiple Actions of the Anthracycline Daunorubicin on Cardiac Ryanodine Receptors

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.

Highly Specific, Conformationally-Dependent Cross-Linking of Lys27 of PLB to SERCA2a in Cardiac SR Vesicles from Humans

Phospholamban (PLB) inhibits SERCA2a, the Ca2+-ATPase of cardiac sarcoplasmic reticulum (SR), by decreasing the apparent Ca2+ affinity of the enzyme. The mechanism of Ca2+ pump inhibition by PLB has been studied by our group using chemical cross-linking agents, which enable PLB-binding interactions with SERCA2a to be measured simultaneously with enzyme inhibition. Previously, cross-linking of canine PLB to SERCA2a was only attainable with Cys-scanning point mutants of PLB. For example, N27C of canine PLB cross-links exclusively to Lys328 of canine SERCA2a with heterobifunctional thiol-to-amine cross-linking agents after co-expression of the two proteins in Sf21 insect cells. Here, we show with SR vesicles prepared from human hearts, that PLB and SERCA2a are cross-linkable using DSG (disuccinimidyl glutarate), a 7.7 Å long, homobifunctional, amine-specific cross-linking agent. Cross-linking of human PLB to SERCA2a takes advantage of the unique Lys residue at position 27 of human PLB, making it susceptible to amine-specific cross-linkers without the need for mutagenesis. This was confirmed by testing SR vesicles prepared from both human and canine ventricles; DSG cross-linked human, but not canine, PLB to SERCA2a. Cross-linking of human PLB to SERCA2a was completely inhibited by either Ca2+ (Ki = 0.50 µM), or the Ca2+ pump inhibitor thapsigargin, but substantially augmented by ATP. This is the first demonstration that PLB binds exclusively to the E2 conformation of SERCA2a in SR vesicles, preferentially the state with bound nucleotide (E2·ATP), and not the state stabilized by thapsigargin (protonated E2). Importantly, similar results were obtained with SR vesicles prepared from both normal and failed human hearts, indicating that PLB-binding interactions with SERCA2a are unchanged in failing myocardium. Studies are in progress to demonstrate with human SR vesicles that Lys27 of PLB cross-links exclusively to Lys328 of SERCA2a.

S-Adenosyl-l-methionine Regulation of the Cardiac Ryanodine Receptor Involves Multiple Mechanisms

Cardiac contraction is triggered by the release of Ca(2+) via the ryanodine receptor (RyR2), a sarcoplasmic reticulum (SR) resident ion channel. RyR2 channel activity is modulated through ligand binding and posttranslational regulatory mechanisms. S-Adenosyl-l-methionine (SAM), the primary methyl group donor for enzyme-mediated methylation of proteins and other biological targets, activates RyR2 via an unknown mechanism. Here we show that the SAM-induced increase in cardiac SR (CSR) vesicle [(3)H]ryanodine binding is unaffected by methyltransferase inhibitors and immunoprecipitation of RyR2 from S-adenosyl-l-[methyl-(3)H]methionine ([(3)H]SAM) pretreated CSR indicates that RyR2 is not a target of SAM-mediated protein methylation. Because SAM contains an adenosine moiety and RyR2 is activated by ATP, we investigated whether SAM exerts its effects through the adenine nucleotide binding sites on the RyR2 channel. In support of this hypothesis, the SAM and ATP concentration dependence of CSR vesicle [(3)H]ryanodine binding virtually overlaps. Furthermore, ryanodine binding assays show that SAM competes with adenine nucleotide activation of RyR2, and the effects of SAM on mean channel open and closed times follow similar trends as those observed for ATP. Interestingly, SAM but not ATP activation of RyR2 was associated with a marked increase in the percent of channel openings to a subconductance level approximately 60% of the maximal single channel conductance. This work highlights the complexity underlying SAM regulation of RyR2 and suggests ligand binding is among the multiple mechanisms responsible for SAM regulation of RyR2.

SAM Regulation of RyR2

RyR2 channel activity is subject to allosteric and posttranslational regulation. S-adenosyl-L-methionine (SAM), the primary methyl group donor for enzyme-mediated methylation of proteins and other biological targets, activates RyR2 via an unknown mechanism. To determine if activation of RyR2 by SAM requires methyltransferase activity, cardiac sarcoplasmic reticulum (CSR) vesicle [3H]ryanodine binding was performed in media containing 150 mM KCl, 20 mM PIPES pH 7.0, 3 μM Ca2+, 1 mM SAM, and 0–1 mM sinefungin, a competitive methyltransferase inhibitor. Sinefungin did not alter CSR vesicle [3H]ryanodine binding, and the SAM-induced increase in [3H]ryanodine binding was not altered by sinefungin. To investigate further whether activation of RyR2 by SAM involves RyR2 methylation, RyR2 was immunoprecipitated from CSR vesicles pretreated for 30 min at 37°C with 3 μCi; 285 nM [3H]SAM plus or minus 500 fold excess cold SAM or 1 mM SAH, an inhibitor of methyltransferase activity, followed by centrifugation through sucrose. Radioactivity incorporated into pretreated CSR and immunoprecipitated RyR2 was determined by liquid scintillation counting. Although the amount of radioactivity incorporated into [3H]SAM pretreated CSR vesicles was reduced from 6.54 to 1.86 and 1.69 pmol [3H]SAM/mg protein in the presence of SAH and excess cold SAM respectively, the amount of radioactivity recovered by immunoprecipitation with anti-RyR was not increased over control (immunoprecipitation without RyR specific antibody). Because SAM contains an adenosine moiety and may activate RyR2 via interaction with the channel's adenine nucleotide binding site(s), the affects of SAM and ATP on RyR2 activity were compared. The SAM and ATP concentration dependence of CSR vesicle [3H]ryanodine binding virtually overlapped with no differences at any concentration tested. This work suggests SAM does not methylate RyR2 and the similarities between ATP- and SAM-induced RyR2 activation support allosteric regulation of RyR2 by SAM.

In Vitro Selection and Characterization of DNA Aptamers Specific for Phospholamban

Calcium transport across the membrane of the sarcoplasmic reticulum (SR) plays an important role in the regulation of heart muscle contraction and relaxation. The sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) 2a is responsible for Ca(2+) up-take by this organelle and is inhibited in a reversible manner by phospholamban, another SR membrane protein. Thus, alleviation of phospholamban-mediated inhibition of SERCA2a is a potential therapeutic option for heart failure and cardiomyopathy. We have now applied the systematic evolution of ligands by exponential enrichment protocol to a library of single-stranded DNA molecules containing a randomized 40-nucleotide sequence to isolate aptamers that bind phospholamban. One of the obtained aptamers, designated Apt-9, was found to specifically bind to the cytoplasmic region of phospholamban in vitro with high affinity (dissociation constant, approximately 20 nM). Apt-9 increased the Ca(2+)-dependent ATPase activity of cardiac SR vesicles but not that of SR vesicles from skeletal muscle in a concentration-dependent manner. It also shifted the Ca(2+) concentration-response curve for this ATPase activity to the left. These effects of Apt-9 were not mimicked by an oligonucleotide with a scrambled version of the Apt-9 sequence. Thus, our results indicate that Apt-9 activates SERCA2a by alleviating the inhibitory effect of phospholamban on this ATPase, and they suggest that phospholamban-specific aptamers warrant further investigation as potential therapeutic agents for heart failure and cardiomyopathy.

Abstract 4625: Dantrolene Inhibits Spontaneous Ca 2+ Release as a Substrate for Catecholaminergic Polymorphic Ventricular Tachycardia (PSVT) by Correcting Defective Inter-Domain Interaction within the Ryanodine Receptor

We previously reported that dantrolene, a therapeutic agent for malignant hyperthermia (MH), prevented abnormal Ca 2+ leak by correction of the defective inter-domain interaction between N-terminal (1– 600) and central (2000 –2500) domains in MH-type ryanodine receptor (RyR). These N-terminal and central domains of the RyR, harbor many mutations associated with MH in RyR1 and CPVT in RyR2. Here, we examined the effect of dantrolene on the Ca 2+ release in CPVT-associated RyR2 R2474S/ + knock-in (KI) mice model. In KI (but not in wild-type:WT) mice (n=6), ventricular tachycardia was observed during or after exercise with treadmill (6/6), which was prevented by pretreatment of dantrolene (20 mg −1 kg −1 day −1 , for 7 days). Cardiac sarcoplasmic reticulum (SR) vesicles were isolated (n=4), then RyR2 was fluorescently labeled with methylcoumarin acetamido (MCA) using DP 2460 – 2495 (DPc10), harboring the CPVT mutation site; R2474S, as a site-directing carrier. Only in KI (but not WT) SR, cAMP (1 μM) reduced stabilizing interactions between N-terminal and central domains (viz. domain unzipping), as assessed by the quenching of the MCA fluorescence by a large-size fluorescence quencher. Dantrolene (1 μM) inhibited the cAMP-induced domain unzipping (in KI) without effect on the cAMP-induced increase in Ser2808 phosphorylation that showed no difference between WT and KI. Using a quartz crystal microbalance technique (a highly sensitive mass-measuring technique), dantrolene was found to specifically bind to the domain 601– 620 of RyR2. In isolated, saponin-permeabilized cardiomyocytes, local Ca 2+ release events were measured using a confocal microscopy with Rhod-2; [Ca 2+ ] was buffered at 30 nM by 0.5 mM EGTA. In KI, the frequency of Ca 2+ sparks (SpF: s −1 · 100 μm −1 ) was much more increased in response to 1μM cAMP (9.7±0.4 in WT; 14.5±0.4 in KI, p<0.01). In the co-presence of dantrolene (1 μM), the increase in SpF in KI was markedly inhibited. In conclusion, dantrolene, by normally restoring the defective inter-domain interaction, seems to correct the hyper-activated channel gating caused by RyR2 mutation, thereby inhibiting spontaneous Ca 2+ release leading to lethal arrhythmia.

Age-dependent effect of oxidative stress on cardiac sarcoplasmic reticulum vesicles

The oxidative stress hypothesis of aging suggests that accumulation of oxidative damage is a key factor of the alterations in physiological function during aging. We studied age-related sensitivity to oxidative modifications of proteins and lipids of cardiac sarcoplasmic reticulum (SR) isolated from 6-, 15- and 26-month-old rats. Oxidative stress was generated in vitro by exposing SR vesicles to 0.1 mmol/l FeSO4/EDTA + 1 mmol/l H2O2 at 37 degrees C for 60 min. In all groups, oxidative stress was associated with decreased membrane surface hydrophobicity, as detected by 1-anilino-8-naphthalenesulfonate as a probe. Structural changes in SR membranes were accompanied by degradation of tryptophan and significant accumulation of protein dityrosines, protein conjugates with lipid peroxidation products, conjugated dienes and thiobarbituric acid reactive substances. The sensitivity to oxidative damage was most pronounced in SR of 26-month-old rat. Our results indicate that aging and oxidative stress are associated with accumulation of oxidatively damaged proteins and lipids and these changes could contribute to cardiovascular injury.

Maurocalcine interacts with the cardiac ryanodine receptor without inducing channel modification

We have previously shown that MCa (maurocalcine), a toxin from the venom of the scorpion Maurus palmatus, binds to RyR1 (type 1 ryanodine receptor) and induces strong modifications of its gating behaviour. In the present study, we investigated the ability of MCa to bind to and modify the gating process of cardiac RyR2. By performing pull-down experiments we show that MCa interacts directly with RyR2 with an apparent affinity of 150 nM. By expressing different domains of RyR2 in vitro, we show that MCa binds to two domains of RyR2, which are homologous with those previously identified on RyR1. The effect of MCa binding to RyR2 was then evaluated by three different approaches: (i) [(3)H]ryanodine binding experiments, showing a very weak effect of MCa (up to 1 muM), (ii) Ca(2+) release measurements from cardiac sarcoplasmic reticulum vesicles, showing that MCa up to 1 muM is unable to induce Ca(2+) release, and (iii) single-channel recordings, showing that MCa has no effect on the open probability or on the RyR2 channel conductance level. Long-lasting opening events of RyR2 were observed in the presence of MCa only when the ionic current direction was opposite to the physiological direction, i.e. from the cytoplasmic face of RyR2 to its luminal face. Therefore, despite the conserved MCa binding ability of RyR1 and RyR2, functional studies show that, in contrast with what is observed with RyR1, MCa does not affect the gating properties of RyR2. These results highlight a different role of the MCa-binding domains in the gating process of RyR1 and RyR2.

Redox Sensitivity of the Ryanodine Receptor Interaction with FK506-binding Protein

The ryanodine receptor (RyR) calcium release channel functions as a redox sensor that is sensitive to channel modulators. The FK506-binding protein (FKBP) is an important regulator of channel activity, and disruption of the RyR2-FKBP12.6 association has been implicated in cardiac disease. In the present study, we investigated whether the RyR-FKBP association is redox-regulated. Using co-immunoprecipitation assays of solubilized native RyR2 from cardiac muscle sarcoplasmic reticulum (SR) with recombinant [(35)S]FKBP12.6, we found that the sulfydryl-oxidizing agents, H(2)O(2) and diamide, result in diminished RyR2-FKBP12.6 binding. Co-sedimentation experiments of cardiac SR vesicles with [(35)S]FKBP12.6 also demonstrated that oxidizing reagents decreased FKBP binding. Matching results were obtained with skeletal muscle SR. Notably, H(2)O(2) and diamide differentially affected the RyR2-FKBP12.6 interaction, decreasing binding to approximately 75 and approximately 50% of control, respectively. In addition, the effect of H(2)O(2) was negligible when the channel was in its closed state or when applied after FKBP binding had occurred, whereas diamide was always effective. A cysteine-null mutant FKBP12.6 retained redox-sensitive interaction with RyR2, suggesting that the effect of the redox reagents is exclusively via sites on the ryanodine receptor. K201 (or JTV519), a drug that has been proposed to prevent FKBP12.6 dissociation from the RyR2 channel complex, did not restore normal FKBP binding under oxidizing conditions. Our results indicate that the redox state of the RyR is intimately connected with FKBP binding affinity.

Role of calmodulin methionine residues in mediating productive association with cardiac ryanodine receptors

Calmodulin (CaM) binds to the cardiac ryanodine receptor Ca2+ release channel (RyR2) with high affinity and may act as a regulatory channel subunit. Here we determine the role of CaM Met residues in the productive association of CaM with RyR2, as assessed via determinations of [3H]ryanodine and [35S]CaM binding to cardiac muscle sarcoplasmic reticulum (SR) vesicles. Oxidation of all nine CaM Met residues abolished the productive association of CaM with RyR2. Substitution of the COOH-terminal Mets of CaM with Leu decreased the extent of CaM inhibition of cardiac SR (CSR) vesicle [3H]ryanodine binding. In contrast, replacing the NH2-terminal Met of CaM with Leu increased the concentration of CaM required to inhibit CSR [3H]ryanodine binding but did not alter the extent of inhibition. Site-specific substitution of individual CaM Met residues with Gln demonstrated that Met124 was required for both high-affinity CaM binding to RyR2 and for maximal CaM inhibition. These results thus identify a Met residue critical for the productive association of CaM with RyR2 channels.

CLIC-2 modulates cardiac ryanodine receptor Ca2+ release channels

We have examined the biochemical and functional properties of the recently identified, uncharacterised CLIC-2 protein. Sequence alignments showed that CLIC-2 has a high degree of sequence similarity with CLIC-1 and some similarity to the omega class of glutathione transferases (GSTO). A homology model of CLIC-2 based on the crystal structure of CLIC-1 suggests that CLIC-2 belongs to the GST structural family but, unlike the GSTs, CLIC-2 exists as a monomer. It also has an unusual enzyme activity profile. While the CXXC active site motif is conserved between CLIC-2 and the glutaredoxins, no thiol transferase activity was detected. In contrast, low glutathione peroxidase activity was recorded. CLIC-2 was found to be widely distributed in tissues including heart and skeletal muscle. Functional studies showed that CLIC-2 inhibited cardiac ryanodine receptor Ca2+ release channels in lipid bilayers when added to the cytoplasmic side of the channels and inhibited Ca2+ release from cardiac sarcoplasmic reticulum vesicles. The inhibition of RyR channels was reversed by removing CLIC-2 from the solution or by adding an anti-CLIC-2 antibody. The results suggest that one function of CLIC-2 might be to limit Ca2+ release from internal stores in cells.

CLIC-2 modulates cardiac ryanodine receptor Ca 2+ release channels

We have examined the biochemical and functional properties of the recently identified, uncharacterised CLIC-2 protein. Sequence alignments showed that CLIC-2 has a high degree of sequence similarity with CLIC-1 and some similarity to the omega class of glutathione transferases (GSTO). A homology model of CLIC-2 based on the crystal structure of CLIC-1 suggests that CLIC-2 belongs to the GST structural family but, unlike the GSTs, CLIC-2 exists as a monomer. It also has an unusual enzyme activity profile. While the CXXC active site motif is conserved between CLIC-2 and the glutaredoxins, no thiol transferase activity was detected. In contrast, low glutathione peroxidase activity was recorded. CLIC-2 was found to be widely distributed in tissues including heart and skeletal muscle. Functional studies showed that CLIC-2 inhibited cardiac ryanodine receptor Ca 2+ release channels in lipid bilayers when added to the cytoplasmic side of the channels and inhibited Ca 2+ release from cardiac sarcoplasmic reticulum vesicles. The inhibition of RyR channels was reversed by removing CLIC-2 from the solution or by adding an anti-CLIC-2 antibody. The results suggest that one function of CLIC-2 might be to limit Ca 2+ release from internal stores in cells.

Stoichiometric Phosphorylation of Cardiac Ryanodine Receptor on Serine 2809 by Calmodulin-dependent Kinase II and Protein Kinase A

Stoichiometric Phosphorylation of Cardiac Ryanodine Receptor on Serine 2809 by Calmodulin-dependent Kinase II and Protein Kinase A