Cardiac Sarcoplasmic Reticulum Membranes Research Articles

Calcium-dependent shifts between phospholamban (PLB) monomers and homo-pentamers reveal the interactions between PLB and SERCA in native cardiac membranes.

Phospholamban monomer (PLB1) binds to the cardiac calcium pump (SERCA2a) and inhibits the enzyme. PLB homopentamers (PLB5) are inactive species, but stable in SDS that correlate well to that in the native membranes. In contrast, SDS separates PLB1 from SERCA2a. Using this feature, we developed assays to probe the constant exchanging PLB1 with SERCA2a, and with PLB5, in native cardiac sarcoplasmic reticulum (SR) membrane vesicles. Dog cardiac SR was pre-incubated at 37°C in 1mM EGTA, allowing PLB1 binding to SERCA2a. Then, 8 µg samples were incubated for 1 min with control buffer, 1mM Ca (free [Ca] = 16.4 µM), 1 mM Ca first and then 5 mM EGTA (free [Ca] = 0.06 µM), or 10µM SERCA2a inhibitor thapsigargin. These reactions were stopped by 1% SDS before SDS-PAGE and immunoblotting to measure PLB5/PLB1 ratios. Addition of Ca significantly increased PLB5/PLB1 to 2.91 ± 0.38 from 1.22 ± 0.08 in the control, Ca-free condition, suggesting that Ca activation of the pump dissociates the whole PLB monomer from inhibitory site on SERCA2a in cardiac membranes. Furthermore, subsequent addition of EGTA reversed Ca-induced increased in PLB5/PLB1 back to 1.38 ± 0.15. Meanwhile, thapsigargin virtually eliminated PLB1 binding to SERCA2a, increasing PLB5/PLB1 to 3.13 ± 0.40. In control experiments, none of these factors affected PLB5/PLB1 of WT-PLB expressed alone in insect cells. Therefore, the detected changes in PLB5/PLB1 must occur in the native SR membranes, reflecting fully reversible processes of Ca-dependent PLB1 association and dissociation from the inhibitory site on SERCA2a, followed by disassembling and assemblage into PLB5, respectively. Such assays will allow further studies to determine the effect of allosteric factors and proteins phosphorylation on the interactions between PLB and SERCA2a directly in native cardiac SR membranes.

Synergistic FRET assays for drug discovery targeting RyR2 channels

A key therapeutic target for heart failure and arrhythmia is the deleterious leak through sarcoplasmic reticulum (SR) ryanodine receptor 2 (RyR2) calcium release channels. We have previously developed methods to detect the pathologically leaky state of RyR2 in adult cardiomyocytes by monitoring RyR2 binding to either calmodulin (CaM) or a biosensor peptide (DPc10). Here, we test whether these complementary binding measurements are effective as high-throughput screening (HTS) assays to discover small molecules that target leaky RyR2. Using FRET, we developed and validated HTS procedures under conditions that mimic a pathological state, to screen the library of 1280 pharmaceutically active compounds (LOPAC) for modulators of RyR2 in cardiac SR membrane preparations. Complementary FRET assays with acceptor-labeled CaM and DPc10 were used for Hit prioritization based on the opposing binding properties of CaM vs. DPc10. This approach narrowed the Hit list to one compound, Ro 90–7501, which altered FRET to suggest increased RyR2-CaM binding and decreased DPc10 binding. Follow-up studies revealed that Ro 90–7501 does not detrimentally affect myocyte Ca2+ transients. Moreover, Ro 90–7501 partially inhibits overall Ca2+ leak, as assessed by Ca2+ sparks in permeabilized rat cardiomyocytes. Together, these results demonstrate (1) the effectiveness of our HTS approach where two complementary assays synergize for Hit ranking and (2) a drug discovery process that combines high-throughput, high-precision in vitro structural assays with in situ myocyte assays of the pathologic RyR2 leak. These provide a drug discovery platform compatible with large-scale HTS campaigns, to identify agents that inhibit RyR2 for therapeutic development.

Negative chronotropism, positive inotropism and lusitropism of 3,5-di-t-butyl-4-hydroxyanisole (DTBHA) on rat heart preparations occur through reduction of RyR2 Ca2+ leak

3,5-Di-t-butyl-4-hydroxyanisole (DTBHA) is considered as an activator of the skeletal muscle sarcoplasmic reticulum (SR) Ca2+-uptake, endowed with antioxidant and L-type Ca2+ channel blocking activities.In this study we assessed the cardiac effects of DTBHA on Langendorff perfused rat hearts, isolated rat atria and rat cardiac SR membrane vesicles, as well as on several SERCA isoforms of membrane preparations. Moreover, in order to clarify its molecular mechanism of action Ca2+ imaging experiments were carried out on HEK293 cells transiently transfected with RyR2 channel. Docking of DTBHA at the rat RyR2 protein was investigated in silico.In Langendorff perfused rat hearts, DTBHA significantly increased, in a concentration-dependent manner, left ventricular pressure and diastole duration, while reducing heart rate and the time-constant of isovolumic relaxation, leaving unaltered coronary perfusion pressure. At the maximum concentration tested (30 µM), it significantly prolonged PQ interval, but left the corrected QT intervals unaffected. In spontaneously beating atria, DTBHA decreased sinus rate in a concentration-dependent manner.DTBHA, at concentrations higher than 10 µM, increased Ca2+ uptake in cardiac SR without affecting Ca2+-dependent ATPase activity assayed on several SERCA isoforms. Moreover, DTBHA antagonized thapsigargin-stimulated Ca2+ leak in cardiac SR and reduced caffeine-induced, RyR2-activated Ca2+ release in RyR2 expressing HEK293 cells. Using computational approaches, DTBHA showed a good affinity outline into binding sites of RyR2 protein.In conclusion, DTBHA behaved like a negative chronotropic, a positive inotropic and a lusitropic agent on rat heart preparations and improved cardiac SR Ca2+ uptake by lowering SR Ca2+ leak.

Acute inhibitory effect of alpha-mangostin on sarcoplasmic reticulum calcium-ATPase and myocardial relaxation.

The benefits of α-mangostin for various tissues have been reported, but its effect on the heart has not been clarified. This study aimed to evaluate the effects of α-mangostin on cardiac function. Using a cardiac sarcoplasmic reticulum (SR) membrane preparation, α-mangostin inhibited SR Ca2+ -ATPase activity in a dose-dependent manner (IC50 of 6.47 ± 0.7 μM). Its suppressive effect was specific to SR Ca2+ -ATPase but not to myofibrillar Ca2+ -ATPase. Using isolated cardiomyocytes, 50 μM of α-mangostin significantly increased the duration of cell relengthening and increased the duration of Ca2+ transient decay, suggesting altered myocyte relaxation. The relaxation effect of α-mangostin was also supported in vivo after intravenous infusion. A significant suppression of both peak pressure and rate of ventricular relaxation (-dP/dt) relative to DMSO infusion was observed. The results from the present study demonstrated that α-mangostin exerts specific inhibitory action on SR Ca2+ -ATPase activity, leading to myocardial relaxation dysfunction.

Phospholamban phosphorylation increases the passive calcium leak from cardiac sarcoplasmic reticulum

Phospholamban (PLN) is a 52 amino acid integral membrane protein of the sarcoplasmic reticulum (SR) that exists in both monomeric and pentameric forms. In its unphosphorylated state, PLN inhibits the SR Ca(2+) ATPase (SERCA). This inhibition is relieved when PLN is phosphorylated as a result of β-adrenergic stimulation of the heart. Consistent with some predictions from molecular models and from functional studies of PLN incorporated into planar lipid bilayers, it has also been postulated that pentameric PLN can also form ion-selective channels. Other molecular models contradict this hypothesis, however. In the work reported here, we used the Ca(2+)-sensitive fluorescent dye Fura-2, to examine the passive Ca(2+) permeability of the SR membrane in vesicles derived from cardiac ventricle. We have found that phosphorylation of PLN by protein kinase A (PKA) leads to an increase in the rate of Ca(2+) leak from Ca(2+)-loaded SR vesicles. This enhanced rate of Ca(2+) leak from the SR is also observed when SR vesicles are incubated with a PLN specific antibody (A1) that mimics phosphorylation of PLN. The ryanodine receptor blocker ruthenium red does not affect the increased rate of Ca(2+) leak from the SR after PLN phosphorylation with PKA or after exposure to A1 antibody, arguing against a possible role of ryanodine receptors in mediating the enhanced leak. Our results are consistent with the hypothesis that phosphorylated PLN forms or regulates a Ca(2+) leak pathway in cardiac SR membranes in situ.

Fosinopril Attenuates the Doxorubicin-induced Cardiomyopathy by Restoring the Function of Sarcoplasmic Reticulum

Fosinopril, an angiotensin-converting enzyme inhibitor, is known to attenuate cardiomyopathy induced by doxorubicin (DOX); however, the mechanisms of this cardioprotection are not fully elucidated yet. In the present study, experimental cardiomyopathy was induced in rats by administration of DOX with or without co-treatment with fosinopril. Fosinopril was utilized on day 1 or 14 of the treatment with DOX to compare efficacies of early versus late co-treatments. We observed that fosinopril attenuated changes induced by DOX (e.g., less increased heart and left ventricular weights, diminished lung congestion and ascites, attenuated LVEDP and LVSP, and less decreased +dP/dt and -dP/dt). Further, fosinopril diminished the levels of markers of cardiac toxicity (i.e., plasma levels and activities of cardiac enzymes and proteins AST, LDH, CPK, cTnI, and BNP). Fosinopril also prevented DOX-induced decreases in Ca(2+) uptake and restored activity of Ca(2+)-stimulated ATPase in left ventricular sarcoplasmic reticulum. We next tested whether the improved Ca(2+) transport activity in sarcoplasmic reticulum was due to modulation of SERCA2 and phospholamban expressions by fosinopril. Fosinopril attenuated the decrease in SERCA2 and phospholamban expressions caused by DOX. In conclusion, cardioprotective effects of fosinopril in the DOX-induced cardiomyopathy appear to be due to its ability to prevent remodeling of the cardiac sarcoplasmic reticulum membrane.

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.

FRET Detection of Calmodulin Binding and Structural Rearrangements Within the Cardiac RyR2 Calcium Release Channel

Calmodulin (CaM) binds to a conserved domain of the ryanodine receptor isoforms expressed in skeletal muscle (RyR1) and cardiac muscle (RyR2) to evoke isoform-specific changes in channel gating. To better understand CaM's interactions with the RyR2 isoform, we are using fluorescence resonance energy transfer (FRET) to define the orientation and kinetics of CaM binding, and to resolve structural rearrangements linked to channel regulation. A FRET donor was targeted to the RyR2 cytoplasmic assembly by preincubating cardiac sarcoplasmic reticulum membranes with a fluorescent-labeled FKBP12.6 (F-FKBP). An acceptor fluorophore was attached within the N-lobe of CaM (F-CaM). A decrease in F-FKBP fluorescence upon addition of F-CaM provided a specific, real-time readout of CaM binding to the RyR2, despite the presence of additional non-RyR CaM targets in the cardiac membranes. FRET demonstrated that the affinity of F-CaM binding to RyR2 was greater in 100 μM than in 30 nM Ca2+. The maximal FRET observed in the presence of saturating [F-CaM] increased as a function of [Ca2+] (30 nM to 1 mM). The Ca2+ dependence of this increase in FRET was similar to the Ca2+ dependence of [3H]ryanodine binding to RyR2 assayed in equivalent media (KCa ∼5 μM). A marked decrease in FRET between FKBP12.6 and CaM was observed when the acceptor was shifted from CaM's N-lobe to CaM's C-lobe. We conclude that CaM binds to the RyR2 in an extended conformation, with its N-lobe oriented nearest to the FKBP12.6 subunit. CaM's conformation and orientation when bound to the RyR2 are therefore similar to what has been demonstrated previously for the RyR1 isoform (Cornea et al., 2009). Ca2+ dependent changes in FRET between FKBP12.6 and CaM may reflect structural changes within the RyR2 linked to channel activation by Ca2+.

Endoplasmic and sarcoplasmic reticulum in the heart

The concept of the presence of sarcoplasmic reticulum (SR) membrane in the heart is widely accepted and has been considered merely to be a different name for the endoplasmic reticulum (ER) in muscle tissues. Cardiac SR membranes are specialized in the regulation of Ca(2+) transport and control of excitation-contraction coupling. By contrast, the ER is responsible for protein synthesis, modification, secretion, lipid and steroid synthesis, and modulation of Ca(2+) signaling. Recent developments have indicated that functional changes in proteins or pathways normally associated with ER and not SR membrane impact cardiac development and pathology. Here, we propose that the SR and ER might be functionally distinct internal membrane compartments in cardiomyocytes.

Inhibition of a cardiac sarcoplasmic reticulum chloride channel by tamoxifen

Anion and cation channels present in the sarcoplasmic reticulum (SR) are believed to be necessary to maintain the electroneutrality of SR membrane during Ca(2+) uptake by the SR Ca(2+) pump (SERCA). Here we incorporated canine cardiac SR ion channels into lipid bilayers and studied the effects of tamoxifen and other antiestrogens on these channels. A Cl(-) channel was identified exhibiting multiple subconductance levels which could be divided into two primary conductance bands. Tamoxifen decreases the time the channel spends in its higher, voltage-sensitive band and the mean channel current. The lower, voltage-insensitive, conductance band is not affected by tamoxifen, nor is a K(+) channel present in the cardiac SR preparation. By examining SR Ca(2+) uptake, SERCA ATPase activity, and SR ion channels in the same preparation, we also estimated SERCA transport current, SR Cl(-) and K(+) currents, and the density of SERCA, Cl(-), and K(+) channels in cardiac SR membranes.

Free radical-induced protein modification and inhibition of Ca2+-ATPase of cardiac sarcoplasmic reticulum.

The effect of oxidative stress on the Ca2+-ATPase activity, lipid peroxidation and protein modification of cardiac sarcoplasmic reticulum (SR) membranes was investigated. Isolated SR vesicles were exposed to FeSO4/EDTA (0.2 micromol Fe2+ per mg of protein) at 37 degrees C for 1 h in the presence or absence of antioxidants. FeSO4/EDTA decreased the maximum velocity of Ca2+-ATPase reaction without a change of affinity for Ca2+ or Hill coefficient. Treatment with radical-generating system led also to conjugated diene formation, loss of sulfhydryl groups, changes in tryptophan and bityrosine fluorescences and to production of lysine conjugates with lipid peroxidation end-products. Lipid antioxidants butylated hydroxytoluene (BHT) and stobadine partially prevented inhibition of Ca2+-ATPase and decrease in tryptophan fluorescence, while the loss of -SH groups and formation of bityrosines or lysine conjugates were completely prevented. Glutathione also partially protected Ca2+-ATPase activity and decreased formation of bityrosine, but it was not able to prevent oxidative modification of tryptophan and lysine. These findings suggest that combination of amino acid modifications, rather than oxidation of amino acids of one kind, is responsible for inhibition of SR Ca2+-ATPase activity.

Aromatic indolinic aminoxyls as antioxidants in cardiac sarcoplasmic reticulum lipid and protein oxidation.

The results presented demonstrate the influence of aromatic indolinic aminoxyls: 1,2-dihydro-2-ethyl-2-phenyl-3H-indole-3-phenylimino-1-oxyl (IA-C2) and 1,2-dihydro-2-octadecyl-2-phenyl-3H-indole-3-phenylimino-1-oxyl (IA-C18) on oxidation of lipids and proteins of cardiac sarcoplasmic reticulum membranes. We have used doxorubicin and t-butyl hydroperoxide as agents inducing oxidative stress in isolated rat cardiac sarcoplasmic reticulum membrane system. Carbonyl groups were measured as the end product of membrane protein oxidation, and thiobarbituric acid reactive substances were assessed as a marker of lipid peroxidation. Inhibition of peroxidation of certain membrane components depends on the length of acyl chain. Aminoxyl IA-C2 inhibits the lipid peroxidation process while IA-C18 is an efficient protector against protein oxidation.

Oligomeric interactions between phospholamban molecules regulate Ca-ATPase activity in functionally reconstituted membranes.

Phospholamban (PLB) is a major target of the beta-adrenergic cascade in the heart, and functions as an endogenous inhibitor of Ca-ATPase transport activity. To identify whether oligomeric interactions between PLB molecules are involved in regulating Ca-ATPase transport activity, we have investigated functional interactions between PLB and the Ca-ATPase in proteoliposomes of purified PLB functionally co-reconstituted with the SERCA2a isoform of the Ca-ATPase isolated from cardiac sarcoplasmic reticulum (SR). The calcium sensitivity of this reconstituted preparation and functional stimulation by cAMP-dependent protein kinase (PKA) are virtually identical to those of the Ca-ATPase in cardiac SR microsomes, ensuring the functional relevance of this reconstituted preparation. Interactions between PLB molecules were measured following covalent modification of the single lysine (i.e., Lys(3)) in PLB isolated from cardiac SR membranes with fluorescein isothiocyanate (FITC) prior to co-reconstitution with the Ca-ATPase. FITC modification of PLB does not interfere with the ability of PLB to inhibit the Ca-ATPase, since FITC-PLB co-reconstituted with the Ca-ATPase exhibits a similar calcium dependence of Ca-ATPase activation to that observed in native SR membranes. Thus, the functional arrangement of PLB with the Ca-ATPase is not modified by FITC modification. Using changes in the anisotropy of FITC-PLB resulting from fluorescence resonance energy transfer (FRET) between proximal PLB molecules to measure the average size and spatial arrangement of FITC chromophores, we find that PLB self-associates to form oligomers whose spatial arrangement with respect to one another is in agreement with earlier suggestions that PLB exists predominantly as a homopentamer. The inability of PKA to activate PLB following covalent modification with FITC permits functional interactions between PLB molecules associated with the Ca-ATPase activation to be identified. A second-order loss of Ca-ATPase activation by PKA is observed as a function of the fractional contribution of FITC-PLB, indicating that PKA-dependent activation of two PLB molecules within a quaternary complex containing the Ca-ATPase is necessary for activation of the Ca-ATPase. We suggest that the requirement for activation of two PLB molecules by PKA represents a physiological mechanism to ensure that activation of the Ca-ATPase following beta-adrenergic stimulation in the heart only occurs above a threshold level of PKA activation.

Inhibition of Ca 2+ release channel (ryanodine receptor) activity by sphingolipid bases: mechanism of action

Sphingosine inhibits the activity of the skeletal muscle Ca 2+ release channel (ryanodine receptor) and is a noncompetitive inhibitor of [ 3H]ryanodine binding (Needleman et al., Am. J. Physiol. 272, C1465-1474, 1997). To determine the contribution of other sphingolipids to the regulation of ryanodine receptor activity, several sphingolipid bases were assessed for their ability to alter [ 3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes and to modulate the activity of the Ca 2+ release channel. Three lipids, N, N-dimethylsphingosine, dihydrosphingosine, and phytosphingosine, inhibited [ 3H]ryanodine binding to both skeletal and cardiac SR membranes. However, the potency of these three lipids and sphingosine was lower in rabbit cardiac membranes when compared to rabbit skeletal muscle membranes and when compared to sphingosine. Like sphingosine, the lipids inhibited [ 3H]ryanodine binding by greatly increasing the rate of dissociation of bound [ 3H]ryanodine from SR membranes, indicating that these three sphingolipid bases were noncompetitive inhibitors of [ 3H]ryanodine binding. These bases also decreased the activity of the Ca 2+ release channel incorporated into planar lipid bilayers by stabilizing a long closed state. Sphingosine-1-PO 4 and C 6 to C 18 ceramides of sphingosine had no significant effect on [ 3H]ryanodine binding to cardiac or skeletal muscle SR membranes. Saturation of the double bond at positions 4–5 decreased the ability of the sphingolipid bases to inhibit [ 3H]ryanodine binding 2–3 fold compared to sphingosine. In summary, our data indicate that other endogenous sphingolipid bases are capable of modulating the activity of the Ca 2+ release channel and as a class possess a common mechanism of inhibition.

Captopril Treatment Improves the Sarcoplasmic Reticular Ca2+Transport in Heart Failure Due to Myocardial Infarction

Although captopril, an angiotensin-converting enzyme (ACE) inhibitor, has been shown to exert a beneficial effect on cardiac function in heart failure, its effect on the status of sarcoplasmic reticulum (SR) Ca2+transport in the failing heart has not been examined previously. In order to determine whether captopril has a protective action on cardiac function, as well as cardiac SR Ca2+-pump activity and gene expression, a rat model of heart failure due to myocardial infarction was employed in this study. Sham operated and infarcted rats were given captopril (2 g/l) in drinking water; this treatment was started at either 3 or 21 days and was carried out until 8 weeks after the surgery. The untreated animals with myocardial infarction showed increased heart weight and elevated left ventricular end diastolic pressure, reduced rates of pressure development and pressure fall, as well as depressed SR Ca2+uptake and Ca2+-stimulated ATPase activities in comparison with the sham control group. These hemodynamic and biochemical changes in the failing hearts were prevented by treatment of the infarcted animals with captopril. Likewise, the observed reductions in the SR Ca2+pump and phospholamban protein contents, as well as in the mRNA levels for SR Ca2+pump ATPase and phospholamban, in the failing heart were attenuated by captopril treatment. These results suggest that heart failure is associated with a defect in the SR Ca2+handling and a depression in the gene expression of SR proteins; the beneficial effect of captopril in heart failure may be due to its ability to prevent remodeling of the cardiac SR membrane.

Nitric oxide synthase in cardiac sarcoplasmic reticulum.

NO. is a free radical that modulates heart function and metabolism. We report that a neuronal-type NO synthase (NOS) is located on cardiac sarcoplasmic reticulum (SR) membrane vesicles and that endogenous NO. produced by SR-associated NOS inhibits SR Ca2+ uptake. Ca2+-dependent biochemical conversion of L-arginine to L-citrulline was observed from isolated rabbit cardiac SR vesicles in the presence of NOS substrates and cofactors. Endogenous NO. was generated from the vesicles and detected by electron paramagnetic resonance spin-trapping measurements. Immunoelectron microscopy demonstrated labeling of cardiac SR vesicles by using anti-neuronal NOS (nNOS), but not anti-endothelial NOS (eNOS) or anti-inducible NOS (iNOS) antibodies, whereas skeletal muscle SR vesicles had no nNOS immunoreactivity. The nNOS immunoreactivity also displayed a pattern consistent with SR localization in confocal micrographs of sections of human myocardium. Western blotting demonstrated that cardiac SR NOS is larger than brain NOS (160 vs. 155 kDa). No immunodetection was observed in cardiac SR vesicles from nNOS knockout mice or with an anti-nNOS mu antibody, suggesting the possibility of a new nNOS-type isoform. 45Ca uptake by cardiac SR vesicles, catalyzed by Ca2+-ATPase, was inhibited by NO. produced endogenously from cardiac SR NOS, and 7-nitroindazole, a selective nNOS inhibitor, completely prevented this inhibition. These results suggest that a cardiac muscle nNOS isoform is located on SR of cardiac myocytes, where it may respond to intracellular Ca2+ concentration and modulate SR Ca2+ ion active transport in the heart.

Anion Permeability and Conduction of Adenine Nucleotides Through a Chloride Channel in Cardiac Sarcoplasmic Reticulum

Cardiac sarcoplasmic reticulum (SR) membrane contains several chloride (Cl-) channels. We have characterized a 116-pS Cl- channel (500 mM cis, 50 mM trans Cl-) in cardiac SR that is activated by protein kinase A-dependent phosphorylation. To understand its function further, we examined the permeation of various anions and adenine nucleotides using the planar lipid bilayer-vesicle fusion technique. This Cl- channel showed a high selectivity to anions and its permeability sequence was Br- > Cl- > I- > NO3- > F-. When all anions were replaced with ATP in the cis solution, channel activity persisted. The conductance was 78 pS with 200 mM ATP and 68 pS with 100 mM ATP. The reversal potentials were +63 mV and +41 mV in 200 mM ATP and in 100 mM ATP, respectively. With 100 mM ADP or AMP in the cis solution, channel activities were also observed. The conductances were 87 pS with 100 mM ADP and 115 pS with 100 mM AMP. The apparent adenine selectivity of this channel was ATP > ADP > AMP, assuming that they exist as divalent anions. These results suggest that the SR Cl- channel in cardiac cells may serve as a transporter for the movement of adenine nucleotides between cytosol and SR lumen.

9-Methyl-7-bromoeudistomin D induces Ca 2+ release from cardiac sarcoplasmic reticulum

9-Methyl-7-bromoeudistomin D (MBED), the most powerful caffeine-like releaser of Ca 2+ from skeletal muscle sarcoplasmic reticulum, induced Ca 2+ release from the cardiac sarcoplasmic reticulum. MBED (5 μM) and caffeine (1 mM) caused rapid Ca 2+ release from the fragmented cardiac sarcoplasmic reticulum in a Ca 2+ electrode experiment. [ 3 H ]MBED bound to a single class of high-affinity binding sites in cardiac sarcoplasmic reticulum membranes ( K d=150 nM). These results suggest that MBED binds to a specific binding site on cardiac sarcoplasmic reticulum membranes to induce Ca 2+ release from the cardiac sarcoplasmic reticulum. Thus, MBED is a useful probe for characterizing Ca 2+ release the channels not only in skeletal sarcoplasmic reticulum but also in cardiac sarcoplasmic reticulum.

High-level coexpression of the canine cardiac calcium pump and phospholamban in Sf21 insect cells.

Phospholamban is a pentameric transmembrane phosphoprotein that regulates the activity of the Ca(2+)-transporting ATPase (SERCA2a) in cardiac sarcoplasmic reticulum. To better understand the structure and function of phospholamban and its mode of regulation of the ATPase, phospholamban and SERCA2a were coexpressed at high levels in Sf21 insect cells using the baculovirus expression system. SERCA2a was expressed as a functionally active Ca2+ pump, accounting for > or = 20% of the total protein in Sf21 cell microsomes. Wild-type phospholamban, as well as phospholamban with different point mutations in the transmembrane region, inhibited both Ca2+ transport and ATP hydrolysis by the recombinant Ca2+ pump. The inhibition of SERCA2a activity was reversed by an anti-phospholamban monoclonal antibody. The phospholamban molecules studied decreased the apparent Ca2+ affinity of the Ca2+ pump, but had no effect on enzyme velocity measured at saturating Ca2+ concentration. Monomeric phospholamban produced by mutations in the leucine/isoleucine zipper domain decreased the apparent Ca2+ affinity the most, giving stronger inhibition of the Ca2+ pump than even wild-type phospholamban. Thus, the baculovirus cell expression system is ideally suited for examining functional interactions between phospholamban and SERCA2a. The results obtained suggest that the phospholamban monomer may be the active species inhibiting the Ca2+ pump in the cardiac sarcoplasmic reticulum membrane.

Glycolytic pathway intermediates activate cardiac ryanodine receptors

During myocardial ischaemia and reperfusion, enhancement of glycolytic activity occurs and this may lead to fluctuating levels of glycolytic intermediates [1–3]. We demonstrate that sugar phosphate intermediates of glycolysis, particularly fructose-1,6-diphosphate (FDP; 100 μM–10 mM), can activate sheep cardiac ryanodine receptor (RyR) channels incorporated into bilayers (open probability (Po) increases up to approximately 0.6) and stimulate [ 3H]ryanodine binding (>200%) to isolated cardiac sarcoplasmic reticulum (SR) membrane vesicles. The relative effectiveness of the sugar phosphates in stimulating [ 3H]ryanodine binding and increasing the Po of the channels was FDP>glucose-1-phosphate (G-1-P)>fructose-6-phosphate (F-6-P)>glucose-6-phosphate (G-6-P). These novel properties of the sugar phosphate compounds indicate that changes in glycolytic flux may influence the release of SR Ca 2+ by modulating RyR channel gating.